Assay for a health state

ABSTRACT

The invention relates to an assay for determining a health state of a subject using a combination of detecting the presence of a virus and detecting the presence of a genomic target or marker indicative of a health state.

TECHNICAL FIELD

The invention relates generally to the detection of the presence of avirus together with the detection of a genomic marker(s) in a sample asan assay for determining the health state of a subject. The invention issuitable as a risk assessment test that has prognostic value.

BACKGROUND ART

A number of procedures are presently available for the detection ofspecific nucleic acid molecules. These procedures typically depend onsequence-dependent hybridisation between the target nucleic acid andnucleic acid probes which may range in length from shortoligonucleotides (20 bases or less) to sequences of many kilobases (kb).

The most widely used method for amplification of specific sequences fromwithin a population of nucleic acid sequences is that of polymerasechain reaction (PCR) (Dieffenbach, C and Dveksler, G. eds. PCR Primer: ALaboratory Manual. Cold Spring Harbor Press, Plainview N.Y.). In thisamplification method, oligonucleotides, generally 20 to 30 nucleotidesin length on complementary DNA strands and at either end of the regionto be amplified, are used to prime DNA synthesis on denaturedsingle-stranded DNA. Successive cycles of denaturation, primerhybridisation and DNA strand synthesis using thermostable DNApolymerases allows exponential amplification of the sequences betweenthe primers. RNA sequences can be amplified by first copying usingreverse transcriptase to produce a complementary DNA (cDNA) copy.Amplified DNA fragments can be detected by a variety of means includinggel electrophoresis, hybridisation with labelled probes, use of taggedprimers that allow subsequent identification (eg by an enzyme linkedassay), and use of fluorescently-tagged primers that give rise to asignal upon hybridisation with the target DNA (eg Beacon and TaqMansystems).

As well as PCR, a variety of other techniques have been developed fordetection and amplification of specific nucleotide sequences. Oneexample is the ligase chain reaction (Barany, F. et al., Proc. Natl.Acad. Sci. USA 88, 189-193, 1991).

Another example is isothermal amplification which was first described in1992 (Walker G T, Little M C, Nadeau J G and Shank D. Isothermal invitro amplification of DNA by a restriction enzyme/DNA polymerasesystem. PNAS 89: 392-396 (1992) and termed Strand DisplacementAmplification (SDA). Since then, a number of other isothermalamplification technologies have been described including TranscriptionMediated Amplification (TMA) and Nucleic Acid Sequence BasedAmplification (NASBA) that use an RNA polymerase to copy RNA sequencesbut not corresponding genomic DNA (Guatelli J C, Whitfield K M, Kwoh DY, Barringer K J, Richmann D D and Gingeras TR. Isothermal, in vitroamplification of nucleic acids by a multienzyme reaction modeled afterretroviral replication. PNAS 87: 1874-1878 (1990): Kievits T, van GemenB, van Strijp D, Schukkink R, Dircks M, Adriaanse H, Malek L, SooknananR, Lens P. NASBA isothermal enzymatic in vitro nucleic acidamplification optimized for the diagnosis of HIV-1 infection. J VirolMethods. 1991 December; 35(3):273-86).

Other DNA-based isothermal techniques include Rolling CircleAmplification (RCA) in which a DNA polymerase extends a primer directedto a circular template (Fire A and Xu SQ. Rolling replication of shortcircles. PNAS 92: 4641-4645 (1995), Ramification Amplification (RAM)that uses a circular probe for target detection (Zhang W, Cohenford M,Lentrichia B, Isenberg H D, Simson E, Li H, Yi J, Zhang D Y. Detectionof Chlamydia trachomatis by isothermal ramification amplificationmethod: a feasibility study. J Clin Microbiol. 2002 January;40(1):128-32.) and more recently, Helicase-Dependent isothermal DNAamplification (HDA), that uses a helicase enzyme to unwind the DNAstrands instead of heat (Vincent M, Xu Y, Kong H. Helicase-dependentisothermal DNA amplification. EMBO Rep. 2004 August; 5(8):795-800.)

Recently, isothermal methods of DNA amplification have been described(Walker G T, Little M C, Nadeau J G and Shank D. Isothermal in vitroamplification of DNA by a restriction enzyme/DNA polymerase system. PNAS89: 392-396 (1992). Traditional amplification techniques rely oncontinuing cycles of denaturation and renaturation of the targetmolecules at each cycle of the amplification reaction. Heat treatment ofDNA results in a certain degree of shearing of DNA molecules, thus whenDNA is limiting such as in the isolation of DNA from a small number ofcells from a developing blastocyst, or particularly in cases when theDNA is already in a fragmented form, such as in tissue sections,paraffin blocks and ancient DNA samples, this heating-cooling cyclecould further damage the DNA and result in loss of amplificationsignals. Isothermal methods do not rely on the continuing denaturationof the template DNA to produce single stranded molecules to serve astemplates from further amplification, but on enzymatic nicking of DNAmolecules by specific restriction endonucleases at a constanttemperature, or unwinding the DNA duplex by the use of helicase enzymes.

The technique termed Strand Displacement Amplification (SDA) relies onthe ability of certain restriction enzymes to nick the unmodified strandof hemi-modified DNA and the ability of a 5′-3′ exonuclease-deficientpolymerase to extend and displace the downstream strand. Exponentialamplification is then achieved by coupling sense and antisense reactionsin which strand displacement from the sense reaction serves as atemplate for the antisense reaction (Walker G T, Little M C, Nadeau J Gand Shank D. Isothermal in vitro amplification of DNA by a restrictionenzyme/DNA polymerase system. PNAS 89: 392-396 (1992). Such techniqueshave been used for the successful amplification of Mycobacteriumtuberculosis (Walker G T, Little M C, Nadeau J G and Shank D. Isothermalin vitro amplification of DNA by a restriction enzyme/DNA polymerasesystem. PNAS 89: 392-396 (1992), HIV-1, Hepatitis C and HPV-16 Nuovo G.J., 2000), Chlamydia trachomatis (Spears P A, Linn P, Woodard D L andWalker G T. Simultaneous Strand Displacement Amplification andFluorescence Polarization Detection of Chlamydia trachomatis. Anal.Biochem. 247: 130-137 (1997).

The use of SDA to date has depended on modified phosphorthioatenucleotides in order to produce a hemi-phosphorthioate DNA duplex thaton the modified strand would be resistant to enzyme cleavage, resultingin enzymic nicking instead of digestion to drive the displacementreaction. Recently, however, several “nickase” enzyme have beenengineered. These enzymes do not cut DNA in the traditional manner butproduce a nick on one of the DNA strands. “Nickase” enzymes includeN.Alw1 (Xu Y, Lunnen K D and Kong H. Engineering a nicking endonucleaseN.Alw1 by domain swapping. PNAS 98: 12990-12995 (2001), N.BstNB1 (MorganR D, Calvet C, Demeter M, Agra R, Kong H. Characterization of thespecific DNA nicking activity of restriction endonuclease N.BstNBI. BiolChem. 2000 November; 381(11):1123-5.) and Mly1 (Besnier C E, Kong H.Converting MlyI endonuclease into a nicking enzyme by changing itsoligomerization state. EMBO Rep. 2001 September; 2(9):782-6. Epub 2001Aug. 23). The use of such enzymes would thus simplify the SDA procedure.

In addition, SDA has been improved by the use of a combination of a heatstable restriction enzyme (Ava1) and Heat stable Exo-polymerase (Bstpolymerase). This combination has been shown to increase amplificationefficiency of the reaction from a 10⁸ fold amplification to 10¹⁰ foldamplification so that it is possible, using this technique, to theamplification of unique single copy molecules. The resultantamplification factor using the heat stable polymerase/enzyme combinationis in the order of 10⁹ (Milla M. A., Spears P. A., Pearson R. E. andWalker G. T. Use of the Restriction Enzyme Ava1 and Exo-Bst Polymerasein Strand Displacement Amplification Biotechniques 1997 24:392-396).

To date, all isothermal DNA amplification techniques require the initialdouble stranded template DNA molecule to be denatured prior to theinitiation of amplification. In addition, amplification is onlyinitiated once from each priming event.

For direct detection, the target nucleic acid is most commonly separatedon the basis of size by gel electrophoresis and transferred to a solidsupport prior to hybridisation with a probe complementary to the targetsequence (Southern and Northern blotting). The probe may be a naturalnucleic acid or analogue such as peptide nucleic acid (PNA) or lockednucleic acid (LNA) or intercalating nucleic acid (INA). The probe may bedirectly labelled (eg with ³²P) or an indirect detection procedure maybe used. Indirect procedures usually rely on incorporation into theprobe of a “tag” such as biotin or digoxigenin and the probe is thendetected by means such as enzyme-linked substrate conversion orchemiluminescence.

Another method for direct detection of nucleic acid that has been usedwidely is “sandwich” hybridisation. In this method, a capture probe iscoupled to a solid support and the target nucleic acid, in solution, ishybridised with the bound probe. Unbound target nucleic acid is washedaway and the bound nucleic acid is detected using a second probe thathybridises to the target sequences. Detection may use direct or indirectmethods as outlined above. Examples of such methods include the“branched DNA” signal detection system, an example that uses thesandwich hybridization principle (1991, Urdea, M. S., et al., NucleicAcids Symp. Ser. 24, 197-200). A rapidly growing area that uses nucleicacid hybridisation for direct detection of nucleic acid sequences isthat of DNA microarrays, (2002, Nature Genetics, 32, [Supplement]; 2004,Cope, L. M., et al., Bioinformatics, 20, 323-331; 2004, Kendall, S. L.,et al., Trends in Microbiology, 12, 537-544). In this process,individual nucleic acid species, that may range from shortoligonucleotides, (typically 25-mers in the Affymetrix system), tolonger oligonucleotides, (typically 60-mers in the Applied Biosystemsand Agilent platforms), to even longer sequences such as cDNA clones,are fixed to a solid support in a grid pattern or photolithographicallysynthesized on a solid support. A tagged or labelled nucleic acidpopulation is then hybridised with the array and the level ofhybridisation to each spot in the array quantified. Most commonly,radioactively- or fluorescently-labelled nucleic acids (eg cRNAs orcDNAs) are used for hybridisation, though other detection systems can beemployed, such as chemiluminescence.

Currently, there is much interest in harnessing molecular methods forthe diagnosis of infectious disease, since such newer methods hold thepromise of sensitive and specific detection of pathogenic organisms. Inthis context, the present invention deals with human papilloma virus(HPV), whose DNA genome exists at the populational level as a variablegene pool with individual HPV types differing both at the nucleotidesequence level as well as in the sizes of their genomes. Detecting andaccurately identifying different HPV types in various clinical samplesvia molecular tests is hampered by the limitations of the variousmolecular tests. In addition, a large number of ‘genotypes’, ‘variants’,‘subtypes’ and ‘types’ exist within the umbrella grouping that definesHPV. For example, there are now over 100 recognized types of HPV some ofwhich are strongly correlated with human disease. The so called high-and medium-risk types are implicated in the progression to cancer andtheir detection via the most accurately available molecular methods isan urgent clinical priority.

The major problem is that detection of HPV alone is not necessarily avery good indicator of progression to cancer. Although CervicalIntraepithelial Neoplasia (CIN) can progress to an invasive form, manylesions either regress or persist but without progressing to carcinoma.Seventy percent of women will clear an HPV infection within two years(1998, Journal of Pediatrics, 132, 277-284; Moscicki, A. B., et al.).However, the finding of CIN and its progression is so variable that evenuntreated, it may return to normalcy or lead to a full blown carcinoma.Approximately one third to one half of CINI and CINII casesspontaneously regress (1990, Australian and New Zealand Journal ofObstetrics and Gynaecology, 30, 1-23., Channen, W et al.), The timetaken to progress from CINI to CINII is of the order of a decade, but insome females can be two decades or more. (2000, Cancer Research, 60,6027-6032., Ylitalo, E et al.).

Viral infection of a cell, tissue or organ can cause the genomemethylation signature of these entities to be altered, as in the case ofEpstein-Barr virus and it's association with gastric carcinoma (2002,American J. Pathology, 160, 787-794, Kang, G. H., et al). Sincemethylation or demethylation is a stable change inherited over many celldivisions, and in some cases, generations of organisms, an alteration ofthe usually stable methylome can be predictive of the pre-cancerous orcancerous state.

It has been challenging to implement reliable and robust DNA-baseddetection systems that recognise all the different HPV types in a singleassay, since not only are there cross hybridization problems betweendifferent HPV genomic types, but the exact classification of whatconstitutes an HPV type is dependent upon genomic sequence similaritieswhich have significant bioinformatic limitations. Thus, while new HPVtypes have been defined as ones where there is less than 90% sequencesimilarity with previous HPV types, finer taxonomic subdivisions aremore problematic to deal with. Thus, a new HPV ‘subtype’ is defined whenthe DNA sequence similarity is in the 90-98% range relative to previoussubtypes. A new ‘variant’ is defined when the sequence similarity isbetween 98-100% of previous variants (1993, Van Rast, M. A., et al.,Papillomavirus Rep, 4, 61-65; 1998, Southern, S. A. and Herrington, C.S. Sex. Transm. Inf. 74, 101-109). This spectrum can broaden further tothe point where variation could be measured based on comparing singlegenomes from single isolated viral particles. In such a case, a‘genotype’ would be any fully sequenced HPV genome that minimallydiffers by one base from any other fully sequenced HPV genome. Thisincludes all cases where a single base at a defined position can existin one of four states, G, A, T or C, as well as cases where the base atthat given position has been altered by deletion, addition,amplification or transposition to another site.

For the above reasons, all the bioinformatic comparisons used in thepresent patent specification application are made relative to the HPV16genome (using positions 1 to 7904 of HPV16 as the standard comparator),and using prior art BLAST methodologies, (1996, Morgenstern, B., et al.,Proc. Natl. Acad. Sci. USA. 93, 12098-12103). The standard HPV ‘type’utilized herein for reference purposes is HPV16 of the Papillomaviridae,a papillomavirus of 7904 base pairs (National Center for BiotechnologyInformation, NCBI locus NC_(—)001526; version NC_(—)001526.1;GI:9627100; references, Medline, 91162763 and 85246220; PubMed 1848319and 2990099).

The difficulties faced by existing HPV detection systems in the contextof disease risk assessment are largely threefold. First limitations ofthe technology systems themselves. Secondly, limitations of thepathological interpretations of diseased cell populations. Thirdly,limitations at the clinical level of assessing disease progression indifferent human populations that are subject to differences in geneticbackground as well as contributing cofactors.

HPV of certain types are implicated in cancers of the cervix andcontribute to a more poorly defined fraction of cancers of the vagina,vulvae, penis and anus. The ring of tissue that is the cervicaltransformation zone is an area of high susceptibility to HPVcarcinogenicity, and assessment of its state from complete cellularnormalcy to invasive carcinoma has been routinely evaluated using visualor microscopic criteria via histological, cytological and molecularbiological methodologies. The early detection of virally-inducedabnormalities at both the viral level and that of the compromised humancell, would be of enormous clinical relevance if it could help indetermining where along a molecular trajectory, from normal to abnormaltissue, a population of cells has reached. However, despite the use ofthe Pap smear for half a century, a solid early risk assessment betweenabnormal cervical cytological diagnoses and normalcy is currently stillproblematical. Major problems revolve around the elusive criteria onwhich to define ‘precancer’, such as the various grades of CervicalIntraepithelial Neoplasia, (CIN1, CIN2 and CIN3) and hence on theclinical decisions that relate to treatment options. Precancerdefinitions are considered by some clinicians to be a pseudo-precise wayin which to avoid using CIN2, CIN3 and carcinoma in situ. There is greatheterogeneity in microscopic diagnoses and even in the clinical meaningof CIN2, (2003, Schiffman, M., J. Nat. Cancer Instit. Monog. 31, 14-19).Some CIN2 lesions have a bad microscopic appearance but willnevertheless be overcome by the immune system and disappear, whereasother lesions will progress to invasive carcinoma. Thus CIN2 isconsidered by some as a buffer zone of equivocal diagnosis although theboundary conditions of such a zone remain controversial. Some cliniciansconsider it to be poor practice to combine CIN2 and CIN3, whereas otherswill treat all lesions of CIN2 or worse. Finally, the literatureindicates that between a third and two thirds of CIN3 assigned womenwill develop invasive carcinoma, but even this occurs in anunpredictable time-dependent fashion, (2003, Schiffman, M., J. Nat.Cancer. Instit. Monog. 31, 14-19; 1978, Kinlen, L. J., et al., Lancet 2,463-465; 1956, Peterson, O. Am. J. Obstet. Gynec. 72, 1063-1071).

The central problem still confronting physicians today is that defininglow grade cytological abnormalities such as atypical squamous cells ofundetermined significance, (ASCUS), or squamous intraepithelial lesions(SILs) is difficult. ‘In fact, ASCUS is not a proper diagnosis butrather is a “wastebasket” category of poorly understood changes’, (1996,Lorincz, A. T., 1996, J. Obstet. Gyncol. Res. 22, 629-636). The wholespectrum of precancerous lesions is difficult to interpret owing tocofactor effects from oral contraceptive use, smoking, pathogens otherthan HPV such as Chlamydia trachomatis and Herpes Simplex Virus type 2,antioxidant nutrients and cervical inflammation, all of which areclaimed to modulate the risk of progression from high grade squamousintraepithelial lesions (HSILs) to cervical cancer (2003, Castellsague,X. J. Nat. Cancer Inst. Monog. 31, 20-28). The introduction of theBethesda system of classification and its revision in 2001 has donelittle to reduce the confusion among clinicians, since it was initiallyfound unhelpful to include koilocytotic atypia with CIN1 into the newercategory of low-grade squamous intraepithelial lesions, (LSILs). Theresult of the introduction of the Bethesda system was that manyclinicians would not carry out colposcopy on koilocytotic atypia, ‘butfelt compelled do so on patients with CIN1’, (1995, Hatch, K. D., Am. J.Obstet. Gyn. 172, 1150-1157). It was clear that although colposcopicexpertise required many years of training, subjective cytologicalcriteria still lead to inconsistencies and non-reproducibilities, (1994,Sherman, M. E., Am. J. Clin. Pathology, 102, 182-187; 1988, Giles, J.A., Br. Med. J., 296, 1099-1102).

The continuing diagnostic hurdle is that vague diagnoses such as‘atypia’ can account for 20% or more of diagnoses in some settings,(1993, Schiffman, M. Contemporary OB/GYN, 27-40). This is illustrated bya test designed specifically to evaluate the level of independentdiagnostic agreement of pathologists on smears that were ‘atypical’. Itwas found that exact agreement between five professional pathologists onan identical set of samples occurred in only 29% of cases, (1994,Sherman, M. E., et al., Am. J. Clin. Pathology, 102, 182-187). The netresult is that cervical cytology continues to have high false negativerates (termed low sensitivity) and high false positive rates, (termedlow specificity). The cytological interpretations of variouspathologists yield a false negative rate of up to 20% or so and a falsepositive rate of up to 15% (1993, Koss, L. G., Cancer, 71, 1406-1412).False positive results lead to unnecessary colposcopic examinations,biopsies and treatments, all of which add to the health care costburden. False negative results lead to potential malpractice law suitswith their associated costs. It was into this arena that moleculardiagnoses of early stages of cervical abnormalities using tests for HPVoffer a less subjective test than cytological ones.

Genomic indicators of a lack of well being in an organism are intimatelytied to changes in genomic methylation status at a number of levels.Dietary supplementation can have unintended and deleterious consequenceson methylation and metabolic well being, (Waterland, R. A., 2003,Molecular and Cellular Biology, 23, 5293-5300) and aberrant methylationof certain genomic promoter regions, such as that of the reelin locus,are implicated in various psychiatric conditions such as schizophrenia(2002, Chen, Y., et al., Nucleic Acids Research, 30, 2930-2939; Miklos,G. L. G., and Maleszka, R., 2004, Nature Biotechnology, 22, 615-621).The single largest area of methylation investigation is in cancerresearch, where both hypermethylation and hypomethylation of genomicregions, is extensively documented (French, S. W., et al., 2002,Clinical Immunology, 103, 217-230; Frigola, J., et al., 2005, HumanMolecular Genetics, 14, 319-326; Belinsky S. A., et al., Nature ReviewsCancer, 4, 1-11). Some of these studies aim at uncovering prognosticbiomarkers (Baker, M., 2005, Nature Biotechnology, 23, 297-304) forindications of cancer, but the biomarker field is riddled withinconsistent results. In addition, rarely are such genomic studiesinterfaced with other sources of perturbations to cells and tissues thatarise from infections with microorganisms and viruses. In addition,cancer genomes generally contain massive genomic upheavals, such aschromosomal aneuploidy, segmental aneuploidy, deletions, amplifications,inversions, translocations and multiple mutations (Duesberg, P., 2004,Cell Cycle, 3, 823-828; Miklos, G. L. G., 2005, Nature Biotechnology,23, 535-537) and the importance of these for early detection, in thecontext of methylation changes, has not yet been very deeply explored,(Vogelstein, B., et al., 2004, Nature Medicine, 10, 789-799; Lucito, R.,et al., 2003, Genome Research, 13, 2291-2305).

Given all the problems and shortcomings outlined above, there is stillcontroversy as regards the clinical impact of DNA methodologiesparticularly in screening for pre-neoplastic lesions. Sensitive earlymolecular prognostic indicators of cellular abnormalities would beextremely valuable. The present inventors have developed new methods,kits and integrated bioinformatic platforms for detecting viruses andgenomic targets for use in determining the health state of anindividual.

DISCLOSURE OF INVENTION

In a general aspect, the present invention relates to an assay fordetermining a health state of a subject using a combination of detectingthe presence of a virus and detecting the presence, absence or status ofa genomic target or marker indicative of a health state. The inventioncan be carried out if required on only one sample in the same test,vessel, or reaction.

In a first aspect, the present invention provides an assay fordetermining a health state of a subject comprising:

(a) treating a sample from a subject with an agent that is capable ofmodifying unmethylated cytosines in nucleic acid, wherein viral nucleicacid and genomic nucleic acid in the sample are treated to formderivative viral nucleic acid and derivative genomic nucleic acid;(b) assaying for the presence of derivative viral nucleic acid in thetreated sample; and(c) determining status of a genomic target in the derivative genomicnucleic acid in the treated sample, wherein the presence of one or moreof derivative viral nucleic acid and the status of the genomic target isindicative of a health state.

In one preferred form, the assay further comprises:

amplifying at least part of the derivative viral nucleic acid andderivative genomic nucleic acid prior to the assaying and determiningsteps.

In a second aspect, the present invention provides an assay fordetermining a health state of a subject comprising:

(a) treating a sample from the subject containing viral nucleic acid andgenomic nucleic acid with an agent that modifies unmethylated cytosineto form derivative viral nucleic acid and derivative genomic nucleicacid having a reduced number of cytosines but having substantially thesame total number of bases as the corresponding untreated viral nucleicacid and untreated genomic nucleic acid;(b) obtaining a virus-specific nucleic acid molecule;(c) obtaining a nucleic acid molecule having a genomic target;(d) testing for the presence of a virus-specific nucleic acid molecule;and(e) determining status of the genomic target in the treated andamplified sample, wherein detection of one or more of the virus-specificnucleic acid molecules and the status of the target is indicative of ahealth state of the subject.

Preferably, the virus-specific nucleic acid molecule and the nucleicacid molecule having a genomic target are obtained by amplifying thederivative viral nucleic acid and the derivative genomic nucleic acid.

In a third aspect, the present invention provides an assay fordetermining a health state of a subject comprising:

(a) treating a sample from a subject with an agent that modifiesunmethylated cytosine to form derivative nucleic acid;(b) providing primers capable of allowing amplification of a desiredviral nucleic acid molecule to the derivative nucleic acid;(c) providing primers capable of allowing amplification of a targetgenomic nucleic acid molecule to the derivative nucleic acid;(d) carrying out an amplification reaction on the derivative nucleicacid; and(e) assaying for the presence of amplified desired viral nucleic acidand amplified target genomic nucleic acid, wherein presence or absenceof one or more amplified products is indicative of a health state of thesubject.

In one preferred form, step (e) comprises assaying for the presence ofan amplified nucleic acid product containing the desired virus-specificnucleic acid molecule, wherein detection of the desired virus-specificnucleic acid molecule is indicative of the presence of the virus in thesample.

In another preferred form, step (e) further comprises assaying for thepresence of an amplified nucleic acid product containing the targetnucleic acid molecule, wherein detection of the target nucleic acidmolecule is indicative of a genomic or gene state in the sample.

In a fourth aspect, the present invention provides an assay fordetermining a health state of a subject comprising:

(a) treating a sample from a subject with a bisulphite reagent underconditions that cause unmethylated cytosines in viral and genomicnucleic acid to be converted to uracil forming derivative viral nucleicacid and derivative genomic nucleic acid;(b) providing primers capable of binding to regions of derivative viralnucleic acid to the sample, the primers being capable of allowingamplification of a desired viral-specific nucleic acid molecule in thederivative viral nucleic acid;(c) providing primers capable of binding to regions of derivativegenomic nucleic acid to the sample, the primers being capable ofallowing amplification of a desired target genomic-specific nucleic acidmolecule in the derivative genomic nucleic acid;(d) carrying out an amplification reaction on the treated sample; and(e) assaying for the presence of an amplified viral nucleic acid productand an amplified genomic nucleic acid target, wherein detection of oneor both of the product and target is indicative of a health state of thesubject.

In a preferred form, the assay further includes:

(f) testing a sample having a virus present to determine the type,subtype, variant or genotype of the virus in the sample.

Amplification can be carried out by any suitable means such as PCR orisothermal amplification.

If the virus has a DNA genome, then treatment with the agent willproduce a derivative nucleic acid. If, however, the virus has an RNAgenome, then the genome can be converted to DNA via a reversetranscriptase methodology. Conversion can be carried out either beforeor after treatment with the agent. Preferably, to ensure that there isno conversion of any other RNA to cDNA, virus specific primers are used.

The subject may be any higher life form that has genomic methylation ata significant frequency. Typically, the present invention is suitablefor animals or humans, and for virally infected plant species.Preferably, the animals are farmed or domesticated mammals, but they maybe wild populations whose health state needs to be monitored. The humancan be a healthy individual or a sick person.

The desired viral nucleic acid molecule can be specific for a virusfamily per se, or a lower taxonomic category, such as genus or species,a type or sub-type, or variant or genotype of virus, whether indicativeof disease or not.

Methylation of some genomic regions typically causes expression ofassociated genes to be turned ‘off’. In certain situations, however,some genes may have associated methylated regions, but gene expressionis still “on”. Thus, in a preferred form, methylation or unmethylationcan be used as a prognostic indicator, rather than for gene expressionper se. It is also possible to block amplification of specific nucleicacid regions so as to determine or target desired methylation states.

The target genomic nucleic acid can be specific for a gene or genes orregulatory region such as a promoter or enhancer, or any coding or noncoding, or static or mobile, region of a genome. Preferably, the targethas a methylation characteristic that is useful for diagnostic orprognostic purposes. This includes mobile or once mobile elements, suchas those exemplified by the LINE family of repetitive DNA sequences(Long INterspersed Elements; also known as Long Interspersed NuclearElements, an abundant retrotransposon family within the human genome;1996, Smit, A. F. A., Current Opinion in Genetics and Development, 6,743-748; 2003, Han, J. S., et al., Nature, 429, 268-274; 2004, Brouha,B., et al., Proc. Natl. Acad. Sci. USA. 100, 5280-5285, and the SINEfamily (Short INterspersed Elements also known as Short InterspersedNuclear Elements; Batzer, M. A., et al.), which together make up nearlyhalf of the human genome. More preferably, the target genomic nucleicacid is indicative of a methylated or unmethylated region of genomicnucleic acid.

Preferably, the assay is repeated with primers specific for a given typeor group of types of virus, wherein the presence of an amplified productis indicative of the type or group of types of virus.

Typically, after amplification, each derivative nucleic acid forms asimplified nucleic acid molecule having a reduced total number ofcytosines compared with the corresponding untreated nucleic acid,wherein the simplified nucleic acid molecule preferably includes anucleic acid sequence specific for the virus or the target.

For double stranded DNA which contains no methylated cytosines, thetreating step results in two derivative nucleic acids, each containingthe bases adenine, guanine, thymine and uracil. The two derivativenucleic acids are produced from the two single strands of the doublestranded DNA. The two derivative nucleic acids have no cytosines butstill have the same total number of bases and sequence length as theoriginal untreated DNA molecule. Importantly, the two derivatives arenot complimentary to each other and form a top and a bottom strand. Oneor more of the strands can be used as the target for amplification toproduce the simplified nucleic acid molecule.

Typically, the simplified nucleic acid sequence specific for the virusdoes not occur naturally in an untreated viral genome.

The virus strain or type can confer a high, medium or low leveloncogenic or other disease status on a given tissue in a particularhuman or animal ethnic lineage. Examples include high risk HPV types 16,18, 45 and 56; medium risk HPV types 30, 31, 33, 35, 39, 51, 52, 58, 59,and 68; and low risk HPV types 6, 11, 42, 43, 44, 53, 54, and 55.

The viruses can be from any of the described families of human viruses,(see; http://www.ncbi.nlm.nih.gov/ICTVdb/lctv/ICD-10.htm) such as fromthe:

-   -   Poxviridae, which includes, cowpox, monkeypox, Vaccinia and        Variola virus. Depending on the virus, these can give rise to        skin and mucous membrane lesions, eczema, and contagious        pustular dermatitis. The various human diseases are summarised        in the 2003, International Statistical Classification of        Diseases and Related Health Problems, (ICD), 10^(th)        revision.(http://www3.who.int/icd/vol1htm2003/navi.htm)    -   Paramyxoviridae which includes Nipah virus, parainfluenza and        Mumps and is associated with various respiratory illnesses,        mumps, meningitis, pancreatitis, encephalitis and measles. Nipah        virus was only recognised first in 1999 and it causes fatal        encephalitis in 70% of infected patients and has an extremely        broad host range including humans, dogs, cats, pigs, horses,        hamsters, bats and guinea pigs. It is a critical threat to        global health and economies (2005, Nature, 436, 401-405;        Negrete, O. A., et al,);    -   Flaviviridae which includes Dengue, Yellow Fever, Hepatitis C        and G and is associated with encephalitis, hepatitis and shock        syndrome. Hep C for example, is an RNA virus with six main        genotypes, is a major cause of chronic liver disease with over        170 million individuals infected worldwide and with no available        vaccine, (2005, Science, 309, 623-626; Lindenbach, B. D., et        al,);    -   Herpesviridae which includes human herpesvirus 1 through 8.        These viruses can give rise to oral infections, ulceration of        the cornea, genital tract infections, meningitis, chickenpox,        pneumonia, shingles, cytomegaloviral mononucleosis and        encephalitis. Human Cytomegalovirus causes severe and fatal        diseases in immunocompromised individuals, including organ        transplant individuals. In addition HHV8 has been implicated as        the causative agent in the AIDS related condition Kaposi        sarcoma. (2003, Nature, 424, 456-461; Wang, X., et al,);    -   Adenoviridae which includes human adenoviruses A through F.        These viruses can give rise to respiratory diseases, infection        of the kidney, conjunctivitis and diarrhoea;    -   Papillomaviridae which includes the Human papilloma virus types        introduced above. These cause viral warts and neoplasms of the        cervix, larynx and bladder;    -   Parvoviridae which includes the B19 virus and gives symptoms of        Rubella without complication;    -   Hepadnaviridae which includes Hepatitis B, and which is        associated with cirrhosis of the liver, and primary        hepatocellular carcinoma;    -   Retroviridae which includes HTLV 1 and 2, HIV 1 and 2,        associated with acute infections and various malignant neoplasms        such as human T-cell leukemias;    -   Reoviridae which includes rotavirus and associated with        diarrhoea, gastroenteritis and upper respiratory tract illness;    -   Filoviridae which includes Marburg and Ebola type viruses and        associated with hemorrhagic fever;    -   Rhabdoviridae which includes vesicular stomatitis and Rabies and        is associated with fever and Rabies;    -   Orthomyxoviridae which includes influenza A, B and C and is        associated with the common cold and pneumonia;    -   Bunyaviridae which includes Crimean-Congo hemorrhagic fever        virus, New York virus, and Hantavirus and is associated with        acute fevers and pulmonary syndromes;    -   Arenaviridae which includes Lassa virus, lymphocytic        choriomeningitis virus and is associated with encephalitis,        meningitis and hemorrhagic fever;    -   Coronaviridae which includes human coronavirus. and is        associated with common cold symptoms and gastrointestinal        symptoms;    -   Picornaviridae which includes human enteroviruses A through D        and polio virus and is associated with bronchitis, meningitis,        and paralysis;    -   Caliciviridae which includes Norwalk-like and Sapporo-like        viruses and is associated with acute gastroenteritis;    -   unassigned “Hepatitis E-like viruses” (HEV) associated with        acute hepatitis;    -   Astroviridae which includes human astrovirus and is associated        with enteritis and gastroenteritis; and    -   Togaviridae which includes Ross River and Rubella and is        associated with encephalitis, leucopenia and rash. Using the        present invention, it is possible that viruses clinically        classified as low risk actually may be high risk in individuals        or animals of a particular genotype or ethnic lineage.

The nucleic acid molecules can be detected by any suitable detectionmeans. Examples include, but not limited to:

providing a detector ligand capable of binding to a region of thenucleic acid molecule and allowing sufficient time for the detectorligand to bind to the region; and

measuring binding of the detector ligand to the nucleic acid molecule todetect the presence of the nucleic acid molecule. It will be appreciatedthat the nucleic acid molecule can be detected by any suitable meansknown to the art.

When a virus-specific nucleic acid molecule has been obtained oridentified for any given virus, probes or primers can be designed toensure amplification of the region of interest in an amplificationreaction. It is important to note that both strands of a treated andthus converted genome, (hereafter termed “derivative”) can be analyzedfor primer design, since treatment or conversion leads to asymmetries ofsequence, (see below), and hence different primer sequences are requiredfor the detection of the ‘top’ and ‘bottom’ strands of the same locus,(also known as the ‘Watson’ and ‘Crick’ strands). Thus, there are twopopulations of molecules, the converted genome as it exists immediatelyafter conversion, and the population of molecules that results after thederivative is replicated by conventional enzymological means (PCR) or bymethods such as isothermal amplification. Primers are typically designedfor the converted top strand for convenience but primers can also begenerated for the bottom strand. Thus, it will be possible to carry outclinical or scientific assays on samples to detect a given type of virusand target in the genome of the organism.

The present invention can use probes or primers that are indicative ofrepresentative types of virus which can be used to determine whether anyvirus is present in a given sample. Further virus type-specific probescan be used to actually detect or identify a given, type, subtype,variant and genotype examples of virus.

The present invention can use probes or primers that are indicative oftargets such as methylation which can be used to determine whether thetarget is present in a given sample. Further target-specific probes canbe used to actually detect or identify targets in the genome.

A real and unexpected advantage of the present invention is that it canbe carried out in the one reaction tube or vessel. Not only is the virusassayed but also genomic targets or targets can be identified. Thecombination of the two test parameters in the one assay allows theassignment of a health state to the subject. The health state can be adisease such as cancer, pre-cancerous state, high risk for diseasestate, and the like. The present invention may be used as an earlyindication of a disease state or potential disease state that will allowearly medical or veterinary or horticultural intervention to preventfurther progression or cure the disease.

The present invention is particularly suitable of detecting viruses thathave been implicated in disease states such as cancer. Examples include,but not limited to human papilloma virus, hepatitis, humanimmunodeficiency virus (HIV), and members of the various families ofviruses described above.

The present invention is particularly suitable of detecting genomictargets in cells that have been implicated in disease states such ascancer. Depending on the disease or health state, potentially any gene,regulatory region or non-coding region in a genome, or its extranuclearor extracellular components, is a potential marker for use in thepresent invention.

The present inventors have obtained data that demonstrate that themethylation state of some genomic regions is indicative of cancerprogression and that the progression is far more reliable in the casewhere the virus is present.

Using clinical samples and cell lines, the present inventors haveexamined methylation patterns in regulatory regions in the vicinity ofnearly 400 human genes and have found over 60 genomic markers that havemethylation changes when in a cancerous state together with the presenceof HPV. Examples include, but not limited to, one or more of thefollowing genomic regions CD14, ENDRB, HIC, RARB1, PGR, SFRS8, TMSB10,ABCG2, MFNG, LAMR1, RAGE, ABL1, CRBP, GPR37, HRK, RARA, SYK, ECE1, MME,TEM, NF2, XIAP, RARRES1, FLI1, HTLF, LDHB, RB1,TGD, CDK4, MMP14, RAB32,BARD1, NF1, LIM2, MMP2, DAB2, BMP6, CDKN1C, DAB2IP, LMNB1, MMP28, HAI2,SOCS1, HIC2, MSH6, RIN2, HMGA1, JUN, S100P*, SRF, VDR, DKK3, KRAS2,PLAU, TNFRSF10B, CDH1, MAC30, DDB2, PAX6, AXL, EIF4A2, SLIT2, RECK,TERC, GATA5, STAT1. Other regions include those shown in Table 8 below.

From the data obtained by the present inventors with HPV, it will beappreciated that each and every disease or health state may haveparticular genomic regions that could be used as markers in the presentinvention. The fact that about approximately 15% of genomic markerstested were only positive (methylated) when the sample was positive forHPV and was derived from a cancerous or pre-cancerous state indicatesthat there may be a large number of possible targets for any givendisease or health state. Given that there are about 25,000 proteincoding loci in the human genome, then 15% rate would predict about 4000possible targets just using the regulatory regions of genes. There wouldbe many more potential targets as specific CpG regions may be methylatedin a particular disease or health state. The invention thereforeencompasses all such possible markers. It will be appreciated that thepresent teaching can be used by a person skilled in the art to determineany useful genomic marker for any disease or health state.

In a fifth aspect, the present invention provides an assay for screeningfor potential cervical cancer in a subject comprising:

(a) treating a sample from the subject with bisulphite reagent underconditions that cause unmethylated cytosines in human papilloma virus(HPV) and genomic nucleic acid to be converted to uracil to formderivative HPV nucleic acid and derivative genomic nucleic acid;(b) providing primers capable of binding to regions of derivative HPVnucleic acid, the primers being capable of allowing amplification of adesired HPV-specific nucleic acid molecule of the derivative HPV nucleicacid;(c) providing primers capable of binding to regions of derivativegenomic nucleic acid, the primers being capable of allowingamplification of a desired genomic-specific nucleic acid molecule of thederivative genomic nucleic acid;(d) carrying out an amplification reaction on the derivative HPV nucleicacid and derivative genomic nucleic acid; and(e) assaying for the presence of an amplified HPV nucleic acid productand an amplified genomic nucleic acid product, wherein detection of oneor both products is indicative of progression to a cervical cancer statein the subject.

In a preferred form, the assay further includes:

(f) testing a sample having the presence of a HPV to determine the type,subtype, variant or genotype of the HPV in the sample.

For HPV16 detection for example, primers capable of binding to thederivative HPV16 viral nucleic acid can be prepared. It will beappreciated that derivative HPV nucleic acid can be determined for allother HPV types by changing all cytosines to uracils in the respectivegenome sequences as carried out for HPV16. Such a conversion depicts theresult of treating the viral nucleic acid in a sample carried out instep a of the assays according to the present invention.

It will be appreciated that other suitable primers or probes can bedevised that are capable of binding to derivative HPV viral nucleic acidfrom other HPV types.

In a sixth aspect, the present invention provides a kit for use in anassay for determining a health state in a subject comprising probes orprimers for a virus-specific nucleic acid molecule and probes or primersfor a genome-specific nucleic acid molecule together with one or morereagents or components for an amplification reaction such as PCR.

The sample includes, but not limited to, swab, biopsy, smear, Pap smear,surface scrape, spatula, and fluid samples, as well as samples fromdifferent storage media such as frozen material, paraffin blocks, glassslides, forensic collection systems and archival material.

Any population of organisms, including animals and humans, as well asplants, will have individuals that are resistant to long term viraleffects. Hence, the combination of viral presence and genomic markersuch as genomic methylation should distinguish whether an organism willhave or is likely to have a disease state. The present invention issuitable for human applications as well as veterinary and otherorganism-based endeavours. For example, the present invention may haveuses in all domestic live-stock where there is sufficient indicationthat genomic methylation occurs, and even as a management tool formonitoring wildlife populations.

In essence, the invention relates to assaying at two levels: one is forthe presence of a virus, and the other is for the presence, absence orstatus of a genomic or nucleic acid target inferred from a particularamplification product or lack of amplification. The target can be amethylation status, nucleic acid sequence, or lack of a nucleic acidsequence, or altered nucleic acid sequence. In some advanced cancers,the genomic target may be completely deleted, and hence the lack of itspresence will be diagnostic. This in itself may be an excellentdiagnostic tool and is covered by the present invention. In addition,many cancers have extra-chromosomal amplicons termed double-minutes, andhence they are not strictly genomic, in that they do not reside in oneof the 46 chromosomes. Similarly, any mitochondrial DNAs, or DNAscontained with cytoplasmic or nuclear organelles are covered by thepresent invention.

The present inventors have developed a combinational technology whichwhen used in a single tube, for example, is far more powerful thaneither technologies used alone in assessing the risk for progression ofa population of cells proceeding towards the cancerous trajectory.Typically the genomic methylation of one or a small number of genomictargets would be done in the one tube. The assay is powerful when thepatient samples containing both viral genomes and host genomic DNA (orRNA) are converted by the use of sodium bisulphite into a simplifiedforms that can be used simultaneously for the detection of both viralpresence and altered host methylation profiles.

Modifying nucleic acid can be the conversion of an unmethylated cytosineto another nucleotide. Preferably, the agent modifies unmethylatedcytosine to uracil which is then replaced as a thymine duringamplification of the derivative nucleic acid. Preferably, the agent usedfor modifying unmethylated cytosine is sodium bisulfite. Other agentsthat similarly modify unmethylated cytosine, but not methylated cytosinecan also be used in the assays of the invention. Examples include, butnot limited to bisulfite, acetate or citrate. Preferably, the agent issodium bisulfite, a reagent, which in the presence of acidic aqueousconditions, modifies cytosine into uracil.

Sodium bisulfite (NaHSO₃) reacts readily with the 5,6-double bond ofcytosine to form a sulfonated cytosine reaction intermediate which issusceptible to deamination, and in the presence of water gives rise to auracil sulfite. If necessary, the sulfite group can be removed undermild alkaline conditions, resulting in the formation of uracil. Thus,potentially all cytosines will be converted to uracils. Any methylatedcytosines, however, cannot be converted by the modifying reagent due toprotection by methylation.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia before thepriority date of each claim of this specification.

In order that the present invention may be more clearly understood,preferred embodiments will be described with reference to the followingdrawings and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows High-Risk HPV testing and HPV typing of various humansamples including; the HeLa cell line; samples from two individuals, (1and 2), designated as having High Grade Squamous IntraepithelialLesions, (designated HSIL-1 and HSIL-2); two individuals with normalcytology, (designated as Normal-1 and Normal-2); and an individual withno cytological indications of a cancerous state, (designated HPV+Nor),but nevertheless inferred to have an HPV infection on the basis ofcellular phenotype as determined by an expert pathologist. A.Determination of High-Risk HPV types in six different samples using aUniversal primer for high-medium risk HPV types; B. Test for HPV usingcomplexity reduced primer sets specific for HPV types 16, 18, 31, 33,35, 39, 45, 51, 52, 56, 58 and 59 and primer sets 42, 43, 44, 53, 54,55, 66, 68, 73, 82, 83 and 84 in the case of the normal tissue sample.

FIG. 2 shows simultaneous detection of Human Genomic DNA and HPV DNA inthe same LBC samples

FIG. 3 shows a representative gel of normal cervical samples amplifiedat 16 different genomic loci, digested with a combination of BstU1 andTaqαI restriction endonuclease, and electrophoresed on an agarose gel.DNA was extracted from liquid based cytology specimens (numbers 28 and29 here), sodium bisulphite modified and amplified with nested primersto genes identified for further analysis. These genes were, from left toright, 1) TEM 2) MME 3) ECE1 4) SYK 5) RARA 6) HRK 7) GPR37 8) CRBP 9)ABL1 10) RAGE 11) LAMR1 12) MFNG 13) ABCG2 14) TMSBIO 15) SFRS8 and 16)PGR.

FIG. 4 shows a representative gel of tumour samples amplified at 16different genomic loci, digested with a combination of BstU1 and TaqαIrestriction endonuclease and electrophoresed on an agarose gel. DNA wasextracted from liquid based cytology specimens (numbers 82, 83, 84, 94,95 and 96 here), sodium bisulphite modified and amplified with nestedprimers to genes identified for further analysis. These genes were, fromleft to right, 1) PGR 2) SFRS8 3) TMSBIO 4) ABCG2 5) MFNG 6) LAMR1 7)RAGE 8) ABL1 9) CRBP 10) GPR37 11) HRK 12) RARA 13) SYK 14) ECE1 15) MMEand 16) TEM.

MODE(S) FOR CARRYING OUT THE INVENTION Definitions

The term “subject” as used herein means refers to an organism that hasgenomic methylation at cytosines. This typically includes Mammalia suchas animals and humans as well as many plant species.

The term “health state” as used herein means the degree to which anindividual differs from that of the general population in terms of areadily definable disease or affliction. For humans this is defined bythe World Heath Organization with its International Classification ofDiseases which is the standard diagnostic classification for all generalepidemiological and health management purposes. In clinical terms thehealth state can be assayed using criteria set out in Harrisons;Principles of Internal Medicine, Braunwald, E et al., eds, McGraw Hill,Medical Publishing Division, 15^(th) and later editions.

The term ‘status of a genomic target’ as used herein can include thepresence or absence of a target nucleic acid (chromosomal orextra-chromosomal), methylation or non-methylation of a genomic target,where that target can be a nucleic acid that is a normal or polymorphicresident of a particular cell type within that population of organisms,or a target that has been introduced via an infectious microorganism orvirus, or a target that has responded by amplification, mobilization,inversion or translocation as a result of an external perturbogen.

The term “modifies” as used herein means the conversion of anunmethylated cytosine to another nucleotide. Preferably, the agentmodifies unmethylated cytosine to uracil which is then replaced as athymine during amplification of the modified nucleic acid.

The term ‘complexity-reduction’ as used herein in the broad sense, meansthat a DNA genome, whether occurring naturally in a eukaryotic,prokaryotic, or viral/viroid life form, or being syntheticallymanufactured, (or if occurring naturally as an RNA virus/viroid genomethen after copying to a cDNA form), which contains the four common basesG, A, T and C, undergoes an increase in the frequency of Ts and adecrease in the frequency of Cs as a result of the bisulphitemodification. This transition changes what was once a normal genome to asequence of polymers, termed the ‘derivatives’, that have no functionalbiological significance and consist of ‘genomic ghosts’.

The term ‘Complexity-reduction’ used herein does not refer to the orderin which bases occur in a derivative population, such as anymathematical complexity difference between a sequence that isATATATATATATAT (SEQ ID NO: 76) versus one of the same length that isAAAAAAATTTTTTT (SEQ ID NO: 77). ‘Complexity-reduction’ as used hereinrefers to an unchanged position of bases in two entities, (one a realgenome and the other a derivative), that are accessed by molecularprobes or primers with both the original genome and its convertedderivative having bases of interest at invariant positions 1 to n. Inthe case of the 3 billion base pair haploid human genome of a particularhuman female, the invariant positions on the ‘top’ strand are defined asbeing from 1 to n, where n is position 3,000,000,000. If in the sequence1 to n, the i^(th) base is a C in the ‘top strand’ of the originalgenome, then the i^(th) base is a U in the converted human derivative.In the case of the 7904 base pair HPV16 viral genome, the invariantpositions are defined as being from 1 to 7904, where n is 7904. If inthe sequence 1 to n, the i^(th) base is a C in the ‘top’ strand of theoriginal genome, then the i^(th) base is a U in the converted HPVderivative. It will be appreciated that when different types of HPVderivatives or types are used for alignments and when those viralderivatives differ in length, then determining the correct i^(th) baserequires careful bioinformatic multiple alignments, as instantiated byMorgenstern, B. et al., Proc. Natl. Acad. Sci. USA. 93, 12098-12103 fornormal multiple genomic alignments.

An example clarifies the consequences of such a conversion process whenapplied to individual viral genomes, or to a mixture of viral genomesthat occurs in a clinical sample containing both human cells and viralgenomes, or parts thereof.

A normal 10 base genomic sequence which is 5′ GGGGAAATTC 3′ (SEQ ID NO:78) (the ‘top’ strand) will have a complementary ‘bottom’ strand that is5′ GAATTTCCCC 3′ (SEQ ID NO: 79). Following denaturation and bisulphitetreatment, the ‘top’ strand becomes 5′ GGGGAAATTU 3′ (SEQ ID NO: 80) andthe ‘bottom’ strand becomes 5′ GAATTTUUUU 3′ (SEQ ID NO: 81). Sincecytosines have been converted to uracils, and uracils are equivalent tothymines in terms of recognition by DNA polymerse machinery ex vivo, thetop strand derivative is essentially 5′ GGGGAAATTT 5′ GGGGAAATTT 3′ (SEQID NO: 82) and the bottom strand derivative is 5′ 5′ GAATTTTTTT 3′ (SEQID NO: 83). Thus an initially normal genome has been converted from onein which the top and bottom strands between them had 5 Cs and 5 Ts, to aderivative population of polymers in which the top and bottom strandsbetween them now have no Cs and 10 Ts. The normal genome has beenreduced from a four base entity to a three base derivative. It has been“complexity-reduced”. In addition, a ‘locus’ in a derivative populationrefers only to positional coordinates within that derivative. Afterbisulphite conversion for example, a locus is stripped of all functionalbiological characteristics at any network level. If it was previouslycoding, regulatory or structural, it is now biological gibberish in bothstrands. A derivative population is thus a collection of functionlesschemical polymers that now represent two non-complementary ghosts of thepreviously complementary strands of a genome that is now informationallyimpotent. Furthermore, the derivatives are unique and do not represent,except by statistical accident, sequences generated by normalevolutionary processes in any cellular, (or viral or viroid), lifeforms.

Probes and Complexity-Reduction

In the formal sense of molecular probes, the present inventors defineherein ‘complexity-reduction’ in terms of the increase in probe length(IPL) that is required to achieve the same specificity and level ofhybridization of a probe to a specific locus, under a given set ofmolecular conditions in two entities of the same size, the first beingthe normal genome and the second being the ‘converted’ entity, (thederivative). For the purposes of molecular utility, IPL is an integerequal to or greater than 1. Each locus remains in the same location inthe normal genome as well as the converted derivative.

In practical terms, ‘complexity-reduction’ can be measured in probelengths. For example, on average, an 11-mer oligonucleotide probe willhave a unique location to which it will hybridize perfectly in a set ofnormal genomes of 4,194,304 bases consisting of the four bases G, A, Tand C (4¹¹ equals 4,194,304). However, once such an initial genome of4,194,304 bases has been converted by the HGS bisulphite methodology,the converted derivative is now T-enriched and is less complex. However,the consequence of this decrease in complexity is that the previouslyunique 11-mer probe no longer has a unique site to which it canhybridize within the complexity-reduced derivative, since other newlyarisen locations of 11 base sequences will have arisen de novo as aconsequence of the bisulphite conversion. These newly arisen sequencesare referred to herein as decoy loci. It will thus now require anapproximately 14-mer probe to find and hybridize to the original locus.In this example, the increase in probe length is approximately from zeroto 3 bases.

Although it may appear counter intuitive, an increased oligonucleotideprobe length may be required to detect the original locus in what is nowa T-enriched derivative. Thus the reduced-complexity of a derivativemeans longer probes may need to be designed for the ‘top’ and ‘bottom’strands of a locus to find the original unique site in the derivative.However, as shown below, the use of Intercalating Nucleic Acid (INA)probes allows for much shorter probes than conventionaloligonucleotides, and so overcomes this requirement for increasedlengths.

‘Complexity-reduction’ as used herein also applies to the differentstructures of probe sequences that can be used in determining thepresence of HPV in a sample. In addition, such probes may havenon-conventional backbones, such as that of PNA, or they can havemodified additions to a backbone such as those described in INA. Thus aderivative is considered to have reduced-complexity irrespective ofwhether the probe has additional components such as intercalatingpseudonucleotides (as in INA). Examples include, but are not limited to,DNA, RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), MNA,altritol nucleic acid (ANA), hexitol nucleic acid (HNA), intercalatingnucleic acid (INA), cyclohexanyl nucleic acid (CNA) and mixtures thereofand hybrids thereof, as well as phosphorous atom modifications thereof,such as but not limited to phosphorothioates, methyl phospholates,phosphoramidites, phosphorodithiates, phosphoroselenoates,phosphotriesters and phosphoboranoates. Non-naturally occurringnucleotides include, but not limited to the nucleotides comprised withinDNA, RNA, PNA, INA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2′-NH)-TNA,(3′-NH)-TNA, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Xylo-LNA, α-D-Ribo-LNA,[3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA,α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA,Bicyclo[4.3.0]amide-DNA, β-D-Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA,2′-R-RNA, α-L-RNA or α-D-RNA, β-D-RNA. In addition non-phosphorouscontaining compounds may be used for linking to nucleotides such as butnot limited to methyliminomethyl, formacetate, thioformacetate andlinking groups comprising amides. In particular nucleic acids andnucleic acid analogues may comprise one or more intercalatorpseudonucleotides (IPN). The presence of IPN is not part of thecomplexity description for nucleic acid molecules, nor is the backbonepart of that complexity, such as in PNA.

By ‘INA’ is meant an intercalating nucleic acid in accordance with theteaching of WO 03/051901, WO 03/052132, WO 03/052133 and WO 03/052134(Human Genetic Signatures Pty Ltd) incorporated herein by reference. AnINA is an oligonucleotide or oligonucleotide analogue comprising one ormore intercalator pseudonucleotide (IPN) molecules.

By ‘HNA’ is meant nucleic acids as for example described by Van Aetschotet al., 1995.

By ‘MNA’ is meant nucleic acids as described by Hossain et al, 1998.

‘ANA’ refers to nucleic acids described by Allert et al, 1999.

‘LNA’ may be any LNA molecule as described in WO 99/14226 (Exiqon).Preferably, LNA is selected from the molecules depicted in the abstractof WO 99/14226. More preferably, LNA is a nucleic acid as described inSingh et al, 1998, Koshkin et al, 1998 or Obika et al., 1997.

‘PNA’ refers to peptide nucleic acids as for example described byNielsen et al, 1991.

The principle of complexity-reduction, defined in terms of probe lengthsand different probe sequences for ‘top’ and ‘bottom’ strands at a locus,is a relative term applicable to different structural or modified probesand primers in different molecular milieu. An example for INAs clarifiesthis relativity. The significant advantages of INAs over the standardoligonucleotide probes are that INAs can be made much shorter thanconventional oligonucleotides and still achieve equivalent hybridizationresults, (INA length<oligonucleotide length). This is due to the highaffinity of INA for complementary DNA owing to the Intercalating PseudoNucleotides, IPNs, that are a structural component of INAs. Thus if itrequires an INA of length X nucleotides, with a given number of IPNs, toachieve successful and specific hybridization to an unconverted genome,it will still require an INA of length>X to hybridize to the same locusin a bisulphite converted genome under the same molecular conditions.

It is also particularly important to note that in the case ofhost-pathogen interactions, (where both viral and host genomes co-existin the same clinical sample but in very different concentrations),‘complexity-reduction’ and the use of INAs introduce new advantageousconditions into hybridization protocols, particularly since INAs have apreference for hybridizing to nucleic acid sequences that areAT-enriched. For example, in a pure solution of unconverted HPV DNA, theapproximate length of a viral probe or primer that is required to findand hybridize to a unique locus in the 7904 base HPV16 genome isapproximately a 6-mer probe/primer, (4⁶ equals 4096 bases). Followingbisulphite treatment to generate a T-enriched HPV derivative, it nowrequires an approximately 8-mer probe or primer to find this uniquelocation, (3⁸ equals 6561 bases) under the same molecular conditions.

However, when two grossly unequally sized genomes are initially presentin a sample, such as the HPV genome of 7904 base pairs and the humangenome of approximately 3,000,000,000 base pairs, and both genomes are‘complexity-reduced’ to their-respective derivatives, the probes orprimers for a unique viral sequence now hybridize to their derivativetargets in a solution that is overwhelmingly dominated by the T-enrichedhuman derivative. If, for example, there was one HPV derivative for eachhuman derivative in the sample, then viral probes or primers arehybridizing to a 3,000,007,904 base pair derivative. Hence assaying fora unique viral sequence now requires approximately 14-mer probes orprimers, to avoid hybridization signals emanating from viral decoy locithat have newly arisen in human sequences.

In addition to ‘complexity-reduction’ issues involving probe and primerlengths, there are also important changes to the kinetics ofhybridization and the ability to detect PCR products when the number ofdegenerate primers used in a PCR reaction is modest. Owing to theextensive genomic variation between HPV types, prior art amplificationshave required the use of a large number of degenerate primers to producerelevant amplified nucleic acid products or amplicons from multiplex PCRreactions. However, the greater the degeneracy in the probe/primer pool,the lower is the concentration of any individual relevant probe orprimer in solution. Such a situation has analogies to the kinetics andfidelity of hybridizations in the driver-tracer reactions carried out oncomplex eukaryotic genomes, and first introduced into the scientificliterature in 1966 by Waring, M. & Britten R. J. Science, 154, 791-794;and in 1968 by Britten, R. J and Kohne D E., Science, 161, 529-540, (andearlier references therein that stem from the Carnegie Institution ofWashington Yearbook reports).

In addition, when HPV PCR primers are in high concentration relative tohuman derivatives, the dominant force in the hybridization reaction isthe HPV primer. For example, if the viral load in a sample is high, (sayof the order of 100,000 HPV genomes to a single human genome), then thekinetics of hybridization of viral primers would be a 100,000 timesfaster than if there were only one HPV derivative per human derivative.In the former case the viral component behaves in solution as if it werea highly repetitive component of a genome. However, in order to detectdifferent HPV types of different risk in a clinical sample by means of asingle PCR reaction, different primers are typically required from eachHPV type necessitating the use of degenerate entities. The net result isthat the primer population can be combinatorially staggering in aconventional multiplex PCR reaction on mixed normal genomes. There canliterally be thousands of different primers competing for hybridizationsites with the net result that PCR amplifications fail, or the amplifiednucleic acid product distribution becomes heavily biased in favor of aparticular HPV type present in the sample. This presents a major problemfor the generation of data from clinical samples in which conventionalunconverted genomes are present.

The present invention of ‘complexity-reduction’, combined with theoptional use of INA probes and primers overcomes many of thedifficulties of these prior art problems.

Primers and Complexity-Reduction

It should be noted that complexity-reduction differs depending uponwhether the population of molecules that has been converted, (thederivatives), remains in the converted state, or is subjected to furtheramplification. In the examples discussed above, the derivativepopulation remained unamplified, as it would exist in a clinical sample.Recall that the top strand (5′ GGGGAAATTC 3′) (SEQ ID NO: 78), and thebottom strand (5′ GAATTTCCCC 3′) (SEQ ID NO: 79), were converted to 5′GGGGAAATTU 3′ (SEQ ID NO: 80) and 5′ GAATTTUUUU 3′ (SEQ ID NO: 81)respectively. Since cytosines have been converted to uracils, anduracils are equivalent to thymines in terms of recognition by DNApolymerse machinery ex vivo, the top strand derivative is essentially 5′GGGGAAATTT 3′ (SEQ ID NO: 82) and the bottom strand derivative is 5′GAATTTTTTT 3′ (SEQ ID NO: 83). However, if the derivative population isnow replicated ex vivo by enzymological means, four distinct derivativepopulations ensue, these being [5′ GGGGAAATTT 3′ (SEQ ID NO: 82)], [5′AAATTTCCCC 3′ (SEQ ID NO: 84)], [5′ AAAAAAATTC 3′ (SEQ ID NO: 85)] and[5′ GAATTTTTTT 3′ (SEQ ID NO: 86)]. These derivatives are indeedcomplexity reduced, but not to the same extent as the originalunreplicated derivatives that exist immediately after conversion. Hencewhen PCR primers are made to the original non-replicated derivativestrands, it is necessary to judiciously decide which amplified nucleicacid products one wishes to examine, as the choice of primers to eitherthe top or bottom strands will influence the output. The differencesbetween dealing with two non-complementary derivative populations thatconstitute the output of a converted genome, versus the four derivativepopulations that exist after replication are not intuitively clears, butcan have important implications for primer design.

Finally, the issue of longer probes or primers that was introducedearlier to formalize and quantitated ‘complexity-reduction’ only assumesrelevance when searching for a unique sequence within a derivativepopulation of molecules. An important foundation of the presentinvention, however, can be the choice of derivative loci that aremaximally similar between virus types, allowing all virus types to beassayed in one initial test, if required. These chosen loci will varydepending upon whether the top or bottom strand derivatives are chosenand such loci will be in different regions in the top strand as comparedto the bottom strand.

The practical importance of the requirement for longer probes andprimers in derivative populations is overshadowed by the practicaladvantages that are gained for virus detection owing to the generationof loci that are rendered more sequence similar by conversion using theHGS bisulphite treatment in the present invention. They are alsoovershadowed by the optional use of INAs that allow for shorter probeand primer molecules than is the case for conventional oligonucleotides.In addition, application of the nested PCR approach to derivativepopulations requires two primers to bind in the same neighbourhood inorder to allow for amplified nucleic acid product production. If one ofthe PCR primers has sequence similarity to a decoy locus that is outsidethe targeted neighbourhood, it is unlikely that the other member of itsprimer pair would also have a decoy locus nearby in the samenon-targeted region. It is even more unlikely that the inner primers ofsuch a nested PCR approach would again have decoy loci in the samenon-targeted region as the first round primers. The probability ofspurious amplification is extremely unlikely.

The term “viral-specific nucleic acid molecule” as used herein means amolecule which has been determined or obtained which has one or moresequences specific to a virus or virus type.

The term ‘taxonomic level of the virus’ as used herein includes type,subtype, variant and genotype. The fluidity of viral genomes isrecognized. Different viral populations may furthermore be polymorphicfor single nucleotide changes or be subject to hyper- or hypo-mutabilityif they reside within certain cancerous cells where normal DNA repairprocesses are no longer functioning.

The term “virus-specific nucleic acid molecule” as used herein means aspecific nucleic acid molecule present in treated or converted viral DNAwhich can be indicative of the virus or virus type.

The term “virus type” as used herein refers to any existing or new viruspopulations where there is less than 90% sequence similarity withpreviously isolated and characterized virus types.

The term “virus subtype” as used herein refers to any existing or newvirus populations where the sequence similarity is in the 90-98% rangerelative to previous subtypes.

The term “virus variant” as used herein refers to any existing or newvirus populations where the sequence similarity is between 98-100% ofprevious variants.

The term “HPV genotype” as used herein is as follows. A genotype is anyfully sequenced HPV genome that minimally differs by one base from anyother fully sequenced HPV genome including whether that single baseexists as either a G, A, T or C, or whether the base at a given positionin the standard comparator, (namely HPV16 from position 1 to position7904) has been altered by deletion, addition, amplification ortransposition to another site. The present inventors compared all otherHPV genotypes relative to the HPV16 standard using prior art BLASTmethodologies.

The term “HPV-specific nucleic acid molecule” as used herein means aspecific nucleic acid molecule present in treated or converted viral DNAwhich can be indicative of the virus or virus type.

The term “HPV type” as used herein refers to any existing or new HPVpopulation where there is less than 90% sequence similarity withpreviously isolated and characterized HPV types, (1993, Van Rast, M. A.,et al., Papillomavirus Rep, 4, 61-65; 1998, Southern, S. A. andHerrington, C. S., Sex. Transm. Inf. 74, 101-109).

The term “HPV subtype” as used herein refers to any existing or new HPVpopulation where the sequence similarity is in the 90-98% range relativeto previous subtypes, (1993, Van Rast, M. A., et al., PapillomavirusRep, 4, 61-65; 1998, Southern, S. A. and Herrington, C. S., Sex. Transm.Inf. 74, 101-109).

The term “HPV variant” as used herein refers to any existing or new HPVpopulation where the sequence similarity is between 98-100% of previousvariants, (1993, Van Rast, M. A., et al., Papillomavirus Rep, 4, 61-65;1998, Southern, A. and Herrington, C. S. Sex. Transm. Inf. 74, 101-109).

The term “HPV genotype” as used herein is as follows. A genotype is anyfully sequenced HPV genome that minimally differs by one base from anyother fully sequenced HPV genome including whether that single baseexists as either a G, A, T or C, or whether the base at a given positionin the standard comparator, (namely HPV16 from position 1 to position7904) has been altered by deletion, addition, amplification ortransposition to another site. The present inventors compared all otherHPV genotypes relative to the HPV16 standard using prior art BLASTmethodologies.

Table 1 provides a list of oligonucleotide primers that have producedHPV-specific products from liquid based cytology samples from humanpatients. All primers are directed to the top strand derivatives of thedifferent HPV types. The designations of the primers are as follows:HPV type-derivative region-primer number as per design below.

For example the top row illustrates primer #1 for HPV16 in the E7derivative region.The HPV-Uni and HPV-HM designations represent the degenerateoligonucleotide primers. Non standard designations are as definedpreviously.

TABLE 1 SEQ ID Designation Sequence NO: HPV16-E7-1TATGTATGGAGATATATTTATATTGT 1 HPV16-E7-2 GTTATGAGTAATTAAATGATAGTTT 2HPV16-E7-3 TAAAACACACAATTCCTAATATAC 3 HPVI6-E7-4CCCATTAATACCTACAAAATCAAC 4 HPV16-E6-1 GAAAGTTATTATAGTTATGTATAGAGT 5HPV16-E6-2 ATTAGAATGTGTGTATTGTAAGTAAT 6 HPV16-E6-3ACTACAATATAAATATATCTCCATAC 7 HPV16-E6-4 AAACTATCATTTAATTACTCATAAC 8HPV16-E4-1 GAATATATTTTGTGTAGTTTAAAGATGATGT 9 HPV16-E4-2GTTTTATATTTGTGTTTAGTAGT 10 HPV16-E4-3 CCTTTTAAATATACTATAAATATAATATTAC 11HPV16-E4-4 CACACAATATACAATATACAATAC 12 HPV18-E7-1GTATGGATTTAAGGTAATATTGTAAGAT 13 HPV18-E7-2 GTATTTAGAGTTTTAAAATGAAATTT 14HPV18-E7-3 AACACACAAAAAACAAAATATTC 15 HPV18-E7-4ACCATTATTACTTACTACTAAAATAC 16 HPV18-E6-1 GATAGTATATAGTATGTTGTATGTT 17HPV18-E6-2 ATTTAGATTTTGTGTATGGAGATAT 18 HPV18-E6-3ATCTTACAATATTACCTTAAATCCATAC 19 HPV18-E6-4 AAATTTCATTTTAAAACTCTAAATAC 20HPV18-E4-1 GGGAATATAGGTAAGTGGGAAGTAT 21 HPV18-E4-2GATTGTAATGATTTTATGTGTAGTATT 22 HPV18-E4-3 AAATAATATATCTCTATAATAATC 23HPV18-E4-4 TTCATTACCTACACCTATCCAATACC 24 HPV56-E7-1GATTTATAGTGTAATGAGTAATTGGATAGT 25 HPV56-E7-2 GGTTATAGTAAGTTAGATAAGT 26HPV56-E7-3 TCCCCATCTATACCTTCAAATAAC 27 HPV56-E7-4CCTATTTTTTTTTCTACAATTAC 28 HPV31-E7-1 GTAATTGATTTTTATTGTTATGAGT 29HPV31-E7-2 GTTATAGATAGTTTAGTTGGATAAGT 30 HPV31-E7-3CTAAATCAACCATTATAATTACAATC 31 HPV31-E7-4 CCTATCTATCTATCAATTACTAC 32HPV33-E7-1 TTTTGTATATGGAAATATATTAGAAT 33 HPV33-E7-2TAGGTGTATTATATGTTAAAGATT 34 HPV33-E7-3 CCTCATCTAAACTATCACTTAATTAC 35HPV33-E7-4 TAACTAATTATACTTATCCATCTAAC 36 HPV35-E7-1AAATAATGTAATAAATAGTTATGTT 37 HPV35-E7-2 GTTGTGTTTAGTTGAAAAGTAAAGAT 38HPV35-E7-3 CCATATATATACTCTATACACACAAAC 39 HPV35-E7-4AAACACACTATTCCAAATATAC 40 HPV39-E7-1 TTAAAGTTTATTTTGTAGGAAATTG 41HPV39-E7-2 GATTTATGTTTTTATAATGAAATATAGT 42 HPV39-E7-3CTAATAAATCCATAAACAACTAC 43 HPV39-E7-4 CATAACAAATTACTAATTTACATTTAC 44HPV58-E7-1 TATTTTGAATTAATTGATTTATTTTGT 45 HPV58-E7-2ATGAGTAATTATGTGATAGTTT 46 HPV58-E7-3 ATACATATACCCATAAACAACTAC 47HPV58-E7-4 TTATTACTATACACAACTAAAAC 48 HPV6-E7-1GATATTTTGATTATGTTGGATATGT 49 HPV6-E7-2 GTTGAAGAAGAAATTAAATAAGAT 50HPV6-E7-3 TACTATCACATCCACAACAACAAATC 51 HPV6-E7-4CTCTAATATCTATTTCTATACACTAC 52 HPV11-E7-1 GTTATGAGTAATTAGAAGATAGT 53HPV11-E7-2 ATTATTAAATATTGATTTGTTGT 54 HPV11-E7-3ATACCTATAATATACTCTACTATAAC 55 HPV11-E7-4 CAAAATTTTATATAATATACCTATC 56HPV42-E7-1 GTATATAGTGGAGAAAGAAATTGGAT 57 HPV42-E7-2GAATAATAAATTAGATGTGTTTTGTGTT 58 HPV42-E7-3 CCAATTATTCATAACAATACAAATC 59HPV42-E7-4 ACTTAATCATCTTCATCTAAAC 60 HPV53-E7-1TAGTGTAYGGGGTTAGTTTGGAAGT 61 HPV53-E7-2 ATTTGATTTATTAATAAGGTGT 62HPV53-E7-3 CTATAATATATTATAAAAATATTAATAC 63 HPV53-E7-4CAATTACTCATAACATTACAAATC 64 HPV-Uni-1 GATGGKGATATGRTDSATRTWGGDTWTGG 65HPV-Uni-2 TAARTATTTWGATTATWTDDRAATG 66 HPV-Uni-3TATTWTAWCCYTAHRCHYWHTAHAACCA 67 HPV-Uni-4 AMAAAHAMHTAATTHYHMMAACAWAYACC68 HPV-HM-1 GATTTDKWDTGTWATGAGTAATT 69 HPV-HML-1 GRKTTDKWDTGTWRKGARTAATT70 HPV-HM-2 RRYRRKTTAGABGADGA 71 HPV-HML-2 RRHRRKTTWGANKWDGA 72 HPV-HM-3YDATACCTWCWMAWWHVDCCAT 73 HPV-HML-3 YDATACCTWHWHHDWHNDCCAT 74 HPV-HML-4ACHHHAAACCAHCCHHWACAHCC 75

Virus Assay

The virus detection used as the first part of assay according to thepresent invention can also be combined with other assays of quitedifferent types for the evaluation of changed cellular status within acell population, for risk assessment underpinned by derangedtranscriptomic, proteomic, metabolite or methylomic networks withininfected cells, for monitoring the progression of an infection and forevaluating a therapeutic regimen such as antiviral therapy.

For example, a molecular assay measuring virus specific nucleic acidmolecules can be combined with:

-   -   assays using pattern recognition and high throughput robotic        imaging technology such as the Multi-Epitope-Ligand-Kartographie        (MELK) system for automated quantitation of fluorescent signals        in tissue sections,    -   assays using light, confocal, transmission or electron        microscopic analyses for Fluorescent In Situ Hybridizations        (FISH), cytological or histological analyses that detect gross        levels of chromosomal disturbance within cells, such as        aneuploidy, or abnormal organelles (in terms of number, type or        morphological appearance),    -   assays using nucleic acid or polypeptide aptamers; Spiegelmers,        (mirror image high-affinity oligonucleotide ligands);        multicoloured nanocrystals (quantum dot bioconjugates), for        ultrasensitive non-isotopic detection of molecules, or        biomarkers for cell surface or internal components;        combinatorial chemistry approaches involving Systematic        Evolution of Ligands by Exponential Enrichment (SELEX) and high        affinity aptamer ligands targeted to different cellular        components,    -   assays using laser-capture of cells or immunomagnetic cell        enrichment technologies, or microsphere-based technologies        interfaced with flow cytometry, or optical barcoding of        colloidal suspensions containing various nucleic acid or        peptide/protein moieties,    -   assays using single cell comparative genomic hybridization aimed        at detecting gross genomic imbalances such as duplications,        deficiencies, transpositions, rearrangements and their        associated in situ technologies,    -   assays reporting on transcriptomic modulations, such as        robogenomic microarray technologies including Serial Analysis of        Gene Expression (SAGE), Total Gene Expression Analyses, (TOGA),        randomly ordered addressable high density fiber-optic sensor        arrays, Massively Parallel Signature Sequencing (MPSS) on        microbeads,    -   assays reporting on proteomic modulations using various        technologies including cellular analyses via protein        microarrays, Matrix Assisted Laser Desorption Ionization-Time of        Flight (MALDI-TOF) methods, Fourier Transformed Ion Cyclotron        Resonance Mass Spectrometry, (FTICR), LC MS-MS and Rapid        Evaporative cooling Mass Spectrometry, (RapEvap MS),    -   assays using Multi Photon Detection (MPD) technologies where the        detection levels approach zeptomole (10⁻²¹) sensitivity,    -   assays using methylomic technologies to interrogate the        methylome of cells from clinical samples to determine the        position of a the cell population along a given trajectory from        normalcy to cervical cancer; preferably to determine the altered        methylation signature of genomic loci in cells which are        affected by viral infection, or immune cells which have been        recruited to the site of infection or inflammation.

Some of the above technologies have been previously evaluated (2001,Miklos and Maleszka, Proteomics, 1, 30-41).

Data Collection, Integration and Management Systems

The data collection and the data management systems for the disclosedmaterial associated with the present invention can be combined withclinical patient data and analysed using specialized algorithmicmethods. Robotic platform management and data collection can beautomatically stored and the collected data combined with an informaticsinfrastructure and software tools that interface with gene ontologies,(GO), with disease ontologies as exemplified by the National Library ofMedicine's Medical Subject Headings (MeSH) thesaurus, the OnlineMendelian Inheritance in Man, (OMIM), or with knowledge databases suchthe Human Genome Mutation Database (HGMD) or PubMed. Software pipelinesthat interface with the latest human genome assemblies and provideaccess to, and downloading of, information from sources such as Genbankand RefSeq, can be combined with assays reporting on the genomic statusof cells that are HPV infected, or that have been influenced by cellsowing to HPV presence elsewhere in the body.

The database infrastructure integrating HPV data with clinical andrelevant bioinformatics data can, for example, utilize a loosely-coupledmodular architecture which facilitates better software engineering anddatabase management. A relational database management system (RDBMS),(such as Postgresq1 version 7.3) is open source and robust, and servesas an example of part of an integrated system to evaluate and betterpredict clinical outcomes in the HPV arena. Additional featuresinvolving web based Graphical User Interfaces (GUI) would allow forintegrated cytological and histological analysis to be combined withmolecular HPV data together with therapeutic and pharmaceutical dataavailable in very diverse formats. The integration of enhanced digitaltechnology for image analysis, remote image sharing by pathologists andautomated visualization systems is envisaged as an integrated part of anautomated molecular kit platform.

Cell Sampling

Virus detection protocols can be implemented on samples from any portionof the body, including samples from pre-blastocyst stages, embryonictissues, perinatal material, cadavers or forensic sources. Preferablythey are from cervicovaginal areas such as the cervix and vagina but canalso be from cutaneous sources. Preferably they are from the cervicaltransformation zone. The samples can be collected using the CervexBrush,Therapak Corp, Irwindale, Calif., USA; Digene Cervical sampler cervicalbrush, Digene Corp. Gaitherburg, Md., USA; a plastic spatula/brushcombination, Cooper Instruments, Hollywood, Fla., USA; or using dacronswabs or any suitable material for obtaining samples from theano-genital area in both male and female patients or by any standardbiopsy procedure such as a needle biopsy. The samples can be placed invarious media, such as PreserveCyte, Cytyc Corp. MA, USA or AutoCytePREP from TriPath Imaging Burlington, N.C., USA. Preferably, initialtests are conducted on Liquid based Cytology, but planar platforms suchas paraffin sections and slides are also suitable.

Kits

The present invention can be implemented in the form of various kits, orcombination of kits and instantiated in terms of manual, semi automatedor fully robotic platforms. In a preferred form, the MethyEasy™ orHighThroughput MethylEasy™ kits (Human Genetic Signatures Pty Ltd,Australia) allow conversion of nucleic acids in 96 or 384 plates using arobotic platform such as EpMotion.

Human Papilloma Virus (HPV)

Mature human papilloma virus DNA is encapsulated within an icosahedralcapsid coat consisting of two virally encoded proteins. The doublestranded circular DNA genome is 7904 base pairs in length for HPV16, butamong the common medium-risk types varies from 7808 base pairs of HPV51to 7942 base pairs of HPV52. The regions of the viral genome arepresented below in the order in which they occur on the circularmolecule. The virus has a non-coding region termed URR followed bynumber of coding regions denoted, E6, E7, E1, E2, E4, E5, L2 and L1.Some viral types may lack a functional E5 region. The E4 region producesmultiple protein products which cause disturbances of the cytoplasmickeratin network, leading to a cytoplasmic “halo effect” termedkoilocytosis. The different HPV types are epitheliotopic and afterinfection can lead to koilocytosis, dyskeratosis, multinucleation,abnormalities such as nuclear enlargement and low grade squamousintraepithelial lesions (SILs), all of these changes applying only tothe cervix. Viral infection and chromosome abnormalities can becorrelated in cervical carcinoma, but the multiparametric changesobserved in neoplastic lesions, and their association with viralinfection, viral gene expression, viral integration, cellulardifferentiation and genomic abnormalities is very poorly understood(1998, Southern, S. A. et al., Sex Transm Inf., 74, 101-109). It is forthis reason that detection of different viral types and their differingeffects in different genetic backgrounds is of such critical importance.

Additionally, although the designation of HPV types into cutaneous andmucosal categories and into high-, medium- and low-risk categories isaccepted in the prior art, these categories exhibit some fraying andoverlap even between the cutaneous and mucosal subcategories of HPV. Forexample HPV7 has been associated with cutaneous warts as well as orallesions. HPV26 has been isolated in the context of generalizedverrucosis as well as anogenital lesions. Furthermore, although HPV6 andHPV11 have been classified as low-risk types, they have been isolatedfrom Buschke-Lowenstein tumors as well as laryngeal and vulvalcarcinomas and condylomata acuminata, (1986, Boshart, M. et al., J.Virology, 58, 963-966; 1992, Rubben, A., et al., J Gen Virol., 73,3147-3153).

Viral integration into the host genome leads to linearization betweenthe E1 and L1 gene regions with retention of the URR, E6 and E7 regions,but with deletion of gene regions such as E1, L1 and L2 and inactivationor deletion of E2. The E6 and E7 regions are generally retained incervical carcinoma whereas E2 protein expression is absent. E2 damagehas been associated with poor prognosis and shortened survival.

Patient Samples

Cell samples were collected by family physicians from the surface of theuterine cervix using a cervix sampling device supplied by CytycCorporation USA. The patients had given consent for the sample to betaken as part of a routine cancer screening program or as a monitoringtest for previous cervical disease. The physicians transferred the cellsfrom the collection device to a methanol/water solution for preservationof the cells and transport to the laboratory for testing. The cellsample was assessed for changes due to pre-cancer or viral infectionsusing routine morphological preparations. A separate aliquot of the cellsample was used for DNA testing as outlined in this specification.

Extraction of DNA

Viral DNA can be obtained from and suitable source. Examples include,but not limited to, cell cultures, broth cultures, environmentalsamples, clinical samples, bodily fluids, liquid samples, solid samplessuch as tissue. Viral DNA from samples can be obtained by standardprocedures. An example of a suitable extraction for paraffin fixedmaterial is as follows. The sample of interest is placed in 400 μl of 7M Guanidinium hydrochloride, 5 mM EDTA, 100 mM Tris/HCl pH 6.4, 1%Triton-X-100, 50 mM Proteinase K (Sigma), 100 μg/ml yeast tRNA. Thesample is thoroughly homogenised with disposable 1.5 ml pestle and leftfor 48 hours at 60° C. After incubation the sample is subjected to fivefreeze/thaw cycles of dry ice for 5 minutes/95° C. for 5 minutes. Thesample is then vortexed and spun in a microfuge for 2 minutes to pelletthe cell debris. The supernatant is removed into a clean tube, dilutedto reduce the salt concentration then phenol:chloroform extracted,ethanol precipitated and resuspended in 50 μl of 10 mM Tris/0.1 mM EDTA.

Surprisingly, it has been found by the present inventors that there isno need to separate the viral DNA from other sources of nucleic acids.The treatment step can be used for an vast mixture of different DNAtypes and yet a viral-specific nucleic acid can be still identified bythe present invention. It is estimated that the limits of detection in acomplex DNA mixtures are that of the limits of standard PCR detectionwhich can be down to a single copy of a target viral nucleic acidmolecule.

High Throughput HPV Assay

The present invention can be used step by step in a high throughputmanner using a 96 well plate in which many samples are simultaneouslytested for HPV. This is illustrated by instructions for a potentialcommercial kit as follows.

Contents of an HPV High Throughput DNA Bisulphite Modification Kit

Component Name Contents Lysis Buffer 1 × 1023 ml Proteinase K 2 × 1 × 2ml Binding Buffer 1 × 35 ml Reagent 1 1 × 20.8 ml Reagent 2 1 × 8 gElution buffer Reagent 3 1 × 725 ml Reagent 4 1 × 7 ml Control Sample 11 × 40 μl Control Sample 2 1 × 1620 μl Control Primers 3A & 3B 2 × 40 μlPurification platePlate 1: 1 × 96 well Incubation plate WashPlate 2:Conversion plate 1 × 96 well ElutionPlate 3: Purification 1 × 96 wellplate Base sealing mat Plate 4: 1 × mat 96 well Wash plate SealingfilmPlate 5: Elution 41 × film 96 well plate Sealing caps 36 × 8 capstrips Plate 6: High Risk HPV 2 × 220 μl 96 well primersplate CarrierDNAPlate 7: HPV 18 × 100 μl 96 well Typing Plate HPV Typing Plate 8:Control 82 × 96 well PlateNB. Individual High-Risk Typing primers sets are available from HumanGenetic Signatures (enquire at <hpv@geneticsignatures.com>)Note: Control Samples/Primers 1, 2, 3A and 3B should be stored at −20°C. upon receipt.

Materials and Equipment Required (not Supplied)

-   -   Either a vacuum manifold or a centrifuge is used as follows:        A vacuum manifold for 96 well plates with a pump to apply at        least −10 in Hg (4.9 psi) pressure. (In-house testing was        carried out using the Biorad Aurum Manifold but other manifolds        may be adapted for use.)

or

A centrifuge with a rotor compatible with a high clearance 96 wellformat plate. (In-house testing was carried out using an Eppendorf5810).

-   -   Heated lid PCR Thermal Cycler compatible for 96 well format 0.2        ml low profile plates    -   Heated lid PCR Thermal Cycler compatible for 384 well format        (for HPV typing)    -   80% isopropanol (molecular biology grade)    -   Water (molecular biology grade)    -   NaOH pellets (Analytical Grade)    -   2×PCR master-mix (Promega Cat# M7505 1000r×n)    -   E-Gel System Mother E-Base™ device (Invitrogen EB-M03)    -   E-gels 96 High-Throughput 2% Agarose (Invitrogen Cat# G7008-02)    -   E-gel Low range marker (Invitrogen Cat# 12373031)    -   Reagent reservoirs×5

Standard Laboratory Equipment (not Supplied)

Multi-channel pipette, up to 1 ml volume (200 μl-1000 μl)

Multi-channel pipette, up to 200 μL volume (20 μl-200 μl)

Multi-channel pipette, up to 10 μL volume (1 μl-10 μl)

Lint-free tissue

Timer

Aerosol barrier tips (10 μl-1000 μl)

Transilluminator

Gel Documentation system

Glison P1000

Gilson P200

Gilson P20

Methods

If using HPV High Throughput DNA Bisulphite Modification Kit for thefirst time, it is highly recommended that the detailed methodology inthe User Guide be read before carrying out the bisulphite conversionmethod.

Using the HPV High Throughput DNA Bisulphite Modification Kit eliminatesthe need for pre-digestion of genomic DNA prior to conversion.

This kit is optimized for starting DNA concentrations from 1 ng up to 4μg of genomic DNA.

Sample Preparation

-   -   Combine the total volume of Reagent 1 to the Reagent 2 bottle        and mix by gentle inversion. Place combined reagents at 72° C.        for 10 minutes or until fully dissolved. Note: Once mixed        Reagents 1 and 2 are stable for up to 1 month at 4° C. in the        dark. All reagents are stable at room temperature for 1 year        from the date of manufacture.    -   Place Binding Buffer in oven or incubator at 72° C. up to 1 hr        before beginning the protocol.    -   Make a fresh 0.3M NaOH solution each time (eg. 0.6 g NaOH in        50.0 ml water).    -   Place the purification plate on top of the wash plate.    -   Add 5 μL of Control Sample 1 to 495 μL of water (molecular        biology grade) and treat in parallel with the test samples.        Control Sample 1 contains an unconverted HPV template that acts        as a process control, and as a sensitivity control. This should        be placed into well H10.    -   Always perform a “No DNA Control” where 500 μL of water is        treated with the rest of the test samples, This should be placed        into well H11.    -   Shake the Liquid Based Sample (PreservCyt®) vial vigorously by        hand to resuspend any sedimented cells and ensure the sample is        well mixed.    -   Transfer 300 μl of the resuspended cells to the appropriate        wells in the purification plate/wash plate combo (Do not use        wells H10, H11 or H12). Make detailed records of which well the        samples were placed into.

Protocol

-   -   Centrifuge the purification plate/wash plate combo at 2000×rcf        for 1 minute and discard the flow through. NB do not discard the        wash plate.    -   Seal the spouts of the purification plate with the base-sealing        mat, making sure all spouts are well sealed. Be sure to align        the cut corner of the sealing mat with the cut corner of the        purification plate at all times.    -   Add 2 ml of Proteinase K to lysis buffer and mix by inversion.    -   Add 100 μl of lysis buffer to each well of the purification        plate (use a 12-channel pipette).    -   Take 10.5 ml of Binding Buffer to a new tube and add 100 μl        Carrier DNA. Mix by inversion and add 100 μl of binding buffer        to each well of the purification plate (use a 12-channel        pipette) then seal the top of the purification plate with the        plate sealing film provided.    -   Incubate at 55° C. for 60 minutes.    -   Carefully remove the base-sealing mat from the purification        plate and quickly place the purification plate on top of the        wash plate. Remove the sealing film from the purification plate        then centrifuge at 2000×rcf for 1 minute and discard the flow        through. NB Do not discard the wash plate.    -   Replace the base-sealing mat onto the purification plate.    -   Add 50 μl of fresh 0.3M NaOH solution to each well of the        Conversion plate and seal the top of the purification plate with        a fresh sealing film (provided).    -   Incubate at 55° C. for 15 minutes.    -   Remove the sealing film and add 220 μl of the combined Reagent 1        and Reagent 2 into each well of the Conversion plate, using a        multi-channel pipette and seal the top of the purification plate        with a fresh sealing film (provided).    -   Incubate the Conversion plate at 55° C. for 3 hours.    -   Following incubation remove the sealing film and add 240 μl of        Binding buffer (Refer to Important Protocol Preparation) to each        well of the Conversion plate, and mix by pipetting, and seal the        top of the purification plate with a fresh sealing film        (provided).    -   Carefully remove the base-sealing mat from the purification        plate and quickly place the purification plate on top of the        wash plate.    -   Remove the sealing film and centrifuge at 2000×rcf for 1 minute        and discard the flow through.    -   Add 500 μl of 80% isopropanol to each well and centrifuge at        2000×rcf for 1 minute at room temperature.    -   Remove the Wash plate, discard the flow-through then replace and        centrifuge at 2,000×rcf for 1 minute at room temperature.    -   Discard the Wash plate and place the Purification plate on top        of the Elution plate ensuring the tips of the Purification plate        are correctly aligned into the Elution plate. Stand plates at        room temperature for 5 minutes.    -   Add 30 μl of Elution Buffer to each sample well of the        Purification plate using a multi-channel pipette, placing the        pipette tip close to the membrane surface without touching it.    -   Incubate at room temperature/1 minute.    -   Repeat above step once more to bring total elution volume to 60        μl    -   Centrifuge the Purification plate/Elution plate combination at        1000×rcf at room temperature/1 minute.    -   Remove the Elution plate and seal with the sealing caps        provided.    -   Incubate the plate in a heated lid PCR machine at 95° C./30        minutes in a heated lid thermo-cycler. Spin briefly before        removing caps.    -   Shake the Liquid Based Sample (PreservCyt®) vial vigorously by        hand to resuspend any sedimented cells and ensure the solution        is homogeneous.    -   Transfer 4 ml of the resuspended cells to a 15 ml Costar        centrifuge tube. If there is less than 4 ml of media transfer        all the material to a 15 ml Costar centrifuge tube and make the        volume to 4 ml with sterile distilled water. A minimum volume of        1 ml sample is required for accurate testing.    -   Centrifuge the tubes in a swing-out bucket rotor at 3000×g/15        minutes.    -   Carefully decant and discard the supernatant without disturbing        the pelleted cellular material.    -   Resuspend the pelleted cells in 200 μl of lysis buffer and mix        well until the solution is homogeneous.    -   Add 20 μl of Proteinase K and incubate to each well of the        incubation plate.    -   Transfer 80 μl of the sample to the Incubation plate (Plate 1)        cover with sealing caps and incubate at 55° C./1 hour.

Protocol Preparation

-   -   Combine the total volume of Reagent 1 to the Reagent 2 bottle        and mix by gentle inversion. Note: Once mixed Reagents 1 and 2        are stable for up to 1 month at 4° C. in the dark. Reagents 1,        2, 3 and 4 are stable at room temperature for 1 year from the        date of manufacture.    -   Make a fresh NaOH solution each time (eg. 1 g NaOH in 8.3 ml        water) and add 5 μl to each well of the Conversion plate (Plate        2).    -   Add 5 μl of Control Sample 1 to 15 μl of water (molecular        biology grade) and treat in parallel with the test samples.    -   Transfer 20 μl of the cell lysate to the Conversion plate        (Plate 2) and mix gently.    -   Seal the Conversion plate (Plate 2) with the sealing film        provided and incubate in an oven at 37° C./15 minutes. After        incubation, centrifuge the plate briefly before removing the        film to precipitate any condensation on the film.    -   Seal the Incubation plate (Plate 1) with sealing caps provided        and store at −20° C.    -   Ensure that Reagent 3 has not formed a solid precipitate. If so,        warm the solution (not higher than 80° C.) and mix.

Centrifugation Protocol

-   -   Add 220 μl of the combined Reagent 1 and Reagent 2 into each        well of the Conversion plate (Plate 2), using a multi-channel        pipette then mix by gentle pipetting and seal the plate with the        8 strip sealing caps provided.    -   Incubate the Conversion plate (Plate 2) in an oven at 55° C./3        hours.        Bisulphite treatment can be carried out in as little as one        hour, however, reducing incubation time can result in regional        non-conversion within the amplicon. Incubation times of less        than 3 hours are therefore not recommended.    -   Following incubation add 240 μl of Reagent 3 (Refer to Important        Protocol Preparation) to each well of the Conversion plate        (Plate 2).    -   Place the Purification plate (Plate 3) on top of the Wash plate        (Plate 4).    -   Transfer the samples from the Conversion plate (Plate 2) to the        corresponding wells of the Purification plate (Plate 3) and        cover with the sealing film provided.    -   Place the Purification plate (Plate 3)/Wash plate (Plate 4)        combination into the centrifuge and spin at 1,000 rcf at room        temperature/4-5 minutes.    -   Discard the flow-through from the Wash plate (Plate 4) then        replace it under the Purification plate (Plate 3). Add 0.8 ml of        80% isopropanol (molecular biology grade) to each well of the        Purification plate (Plate 3).    -   Centrifuge at 1,000 rcf at room temperature/1 minute.    -   Remove the Wash plate (Plate 4), discard the flow-through, then        replace and centrifuge at 1,000 rcf/2 minutes at room        temperature.    -   Place the Purification plate (Plate 3) on top of the Elution        plate (Plate 5) ensuring the tips of the Purification plate        (Plate 3) are positioned within the appropriate wells of the        Elution plate (Plate 5).    -   Add 50 μl of Reagent 4 to each sample well of the Purification        plate (Plate 3) using a multi-channel pipette, placing the        pipette tip close to the membrane surface without touching it.    -   Incubate at room temperature/1-2 minute.    -   Centrifuge the Purification plate (Plate 3)/Elution plate        (Plate 5) combination at 1,000 rcf at room temperature/1 minute.    -   Remove the Elution plate (Plate 5) and seal with the sealing        caps provided.    -   Incubate the plate in a heated lid PCR machine at 95° C./30        minutes        The DNA samples are now converted and ready for PCR        amplification. After incubation centrifuge the plate briefly to        remove any condensation from the sealing caps.

Internal Control PCR Reaction

Genomic DNA and control PCR primers have been provided to allow for easytroubleshooting. Control Samples 1 (purple) and 2 (green) are providedas process controls. Control Sample 1 is untreated DNA with sufficientmaterial provided for 8 conversion reactions. Control Sample 2 isbisulphite treated DNA with sufficient material provided for 20 PCRamplifications. Control Primers 3A (yellow) and 3B (red) are PCR primersand may be used to check the integrity of the recovered DNA (sufficientfor 20 PCR amplifications provided).‘Nested’ PCR primers are used to further improve the sensitivity of thedetection that is achieved with HPV High Throughput DNA BisulphiteModification Kit. The control primers are conventional bisulphite PCRprimers and have been optimised for two rounds of PCR amplification. Theuse of these PCR primers for single round PCR is not recommended as inmost cases no visible amplicon band will be seen following agarose gelelectrophoresis.Note: This protocol is based on the use of a heated-lid thermal cycler.If a heated-lid thermal cycler is unavailable, overlay reactions withmineral oil.

Control Reactions:

Control Sample 1 (purple) contains untreated HPV genomic DNA (50 ng/μl)

Control Sample 2 (green) contains bisulphite treated HPV human DNA (20ng/μl)

Control Primers 3A (yellow) contains First round PCR primers

Control Primers 3B (red) contains Second round PCR primers

Control PCR

Control Primers 3A (First round PCR primers) and Control Primers 3B(Second round PCR primers) are validated ‘nested’ primers withsufficient volume supplied for up to 20 control PCR reactions. Theseprimer samples have been supplied to facilitate the trouble-shootingprocess if required, and may also be used to assess the quality of yourmodified DNA.Note: The Second round PCR Reactions may be prepared in parallel withthe First round PCR Reactions and frozen until required.

High-Risk PCR Amplification First Round Amplification

-   -   For each reaction, add 12.5 μl of PCR Master Mix (for example,        Promega Master Mix) and 9.5 μl water (molecular biology grade)        in the High-Risk PCR plate provided. If you are setting up 96        samples combine 1.25 ml Master mix, 850 μl of water and 200 μl        of primer mix in an appropriate tube and mix well. Then using a        multi channel pipette add 23 μl of the reaction mix to each well        in the High-Risk HPV plate (Plate 6) provided.    -   Add 2 μl of Control Primers 3A to the appropriate well to        control well H10 and H11.    -   Add 2 μl of the required modified DNA from the Elution plate        (Plate 5) to the High-Risk HPV plate (Plate 6) provided and 2 μl        of Control Sample 2 to well H11 then store the remainder at        −20° C. for subsequent HPV typing (see below for High-Risk plate        lay-out).    -   Run the following PCR program.

95° C./3 min  1 cycle 95° C./1 min 30 cycles 42° C./2 min 60° C./2 min65° C./10 min  1 cycle

Second Round Amplification

-   -   Add 2 μl of the first round amplified DNA to second round mixes,        prepared exactly the same as for the first round amplifications.    -   Run the following PCR program

95° C./3 min  1 cycle 95° C./1 min 30 cycles 42° C./2 min 60° C./2 min60° C./10 min  1 cycle

Electrophoresis

-   -   Remove the 96 well 2% E-gel from the foil wrapper and remove the        red 96 well comb.    -   Add 10 μl of sterile water to each well of the gel using a        multi-channel pipette.    -   Add 10 μl of DNA marker to the marker wells.    -   Transfer 10 μl of amplified product to each well of the E-gel        using a multichannel pipette.    -   Set the E-base for 5-7 minutes and press pwr/prg.    -   Record the results using an UV transilluminator and gel        documentation software.

HPV Typing First Round Amplification

The High-Risk Typing plate (Plate 8) contains strain specific primersdirected against the following high-risk HPV types: 16, 18, 31, 33, 35,39, 45, 51, 52, 56, 58, 59 and 68. There is sufficient DNA remaining inthe Elution plate (Plate 5) to type each sample for all high-riskstrains.

-   -   Remove the Elution plate (Plate 5) from the −20° C. freezer.    -   Any samples positive by the high-risk universal amplification        can now be typed using the strain specific primers (see below        for typing plate set-up)    -   For each reaction, add 12.5 μl of PCR Master Mix (for example,        Promega Master Mix) and 8.5 μl water into each well of the PCR        plate provided. If you have 6 samples to type add 1187.5 μl of        Master Mix and 807.5 μl of water into an appropriate tube, mix        well then add 21 μl to each well of the HPV Typing plate        (Plate 7) as indicated below.    -   Add 2 μl of the appropriate primer set to each well as indicated        below.    -   If the typing is being carried out in 384 well format and 24        samples are available for typing add 4.5 ml of Master Mix and        3.42 ml of water into an appropriate tube, mix well then add 21        μl to each well of the 384 well plate as indicated below. Then        add 2 μl of the appropriate primer set to each well as indicated        below.    -   Add 2 μl of High-Risk positive sample (from Elution plate,        Plate 5) to the appropriate wells of the typing plate.

Set up sufficient tubes for each of your samples and a ‘no template’(negative) control.

Run the following PCR program.

95° C./3 min  1 cycle 95° C./1 min 30 cycles 45° C./2 min 65° C./2 min65° C./10 min  1 cycle

Second Round Amplification

-   -   Add 2 μl of the First round amplified DNA to Second round mixes,        prepared exactly the same as for the First round amplifications.    -   Run the following PCR program

95° C./3 min  1 cycle 95° C./1 min 30 cycles 45° C./2 min 65° C./2 min65° C./10 min  1 cycle

Electrophoresis

-   -   Remove the 96 well 2% E-gel from the foil wrapper and remove the        red 96 well comb.    -   Add 10 μl of sterile water to each well of the gel using a        multi-channel pipette.    -   Add 10 μl of DNA marker to the marker wells.    -   Transfer 10 μl of amplified product to each well of the E-gel        using a multichannel pipette.    -   Set the E-base for 5-7 minutes and press run.    -   Record the results using an UV transilluminator and gel        documentation software.    -   The sample has now been typed.

Troubleshooting

PROBLEMS POSSIBLE SOLUTIONS No PCR product was PCR has failed - makesure all the found for any sample components were added to the tube andthat the PCR cycle was correct. Confirm that the polymerase is withinits storage date and that it retains its activity. No PCR product wasfound Modification has failed - check that the for any sample exceptNaOH solution was fresh and that for Control Sample 2 combined Reagent #1 and Reagent 2 was no older than 4 weeks. Make sure that all the stepsin the modification and clean up protocols were followed. DNA wasdegraded during modification - check that all reagents and tubes usedduring the procedure were of molecular biology quality (ie DNase free).Modification was incomplete. Return the samples to 95° C. for a further15 minutes. Sample DNA was degraded before modification- check that theDNA has been stored/handled correctly. PCR products were present Checkthat the DNA concentration is not only in the control reactions toodilute. Check that the PCR-grade water and not the template was added tothe negative control. PCR products were present Make sure that the PCRis being set up in in all the lanes including a separate area withdedicated reagents the ‘no-template’ and equipment to prevent cross(negative) control contamination.

Bisulfite-treated HPV DNA from sources, when amplified using genomicallysimplified primers, be they oligonucleotides or modified nucleic acidssuch as INAs provide an unsurpassed detection system for finding HPV ofany type within a sample, be that sample from human clinical material orat another extreme from an environmental source. The present inventionhas been developed for a clinically relevant virus (HPV) believed to becausative for a human cancer.

The practical implications of the detection assay according to thepresent invention can be varied. While the principles described indetail above have been demonstrated using PCR for amplification,readouts can be engaged via any methodology known in the art. With thecurrent emphasis on microarray detection systems; one would be able todetect a great diversity of HPV using genomically simplified DNA sincethe bisulfite treatment reduces the genomic complexity and hence allowsfor more types of HPV to be tested on microarrays with a smaller numberof detectors (features).

In summary, the HGS genomically simplified primer methodology yieldsconsistent data sets that has been correlated with the clinicalphenotypes of a number of patients.

Bisulphite Treatment

An exemplary protocol for effective bisulphite treatment of nucleic acidis set out below. The protocol results in retaining substantially allDNA treated. This method is also referred to herein as the Human GeneticSignatures (HGS) method. It will be appreciated that the volumes oramounts of sample or reagents can be varied.

Preferred method for bisulphite treatment can be found in U.S. Ser. No.10/428,310 or WO 2004/096825 (PCT/AU2004/000549) in the name of HumanGenetic Signatures Pty Ltd, incorporated herein by reference.

To 2 μg of DNA, which can be pre-digested with suitable restrictionenzymes if so desired, 2 μl ( 1/10 volume) of 3 M NaOH (6 g in 50 mlwater, freshly made) was added in a final volume of 20 μl. This stepdenatures the double stranded DNA molecules into a single stranded form,since the bisulphite reagent preferably reacts with single strandedmolecules. The mixture was incubated at 37° C. for 15 minutes.Incubation at temperatures above room temperature can be used to improvethe efficiency of denaturation.

After the incubation, 208 μl 2 M Sodium Metabisulphite (7.6 g in 20 mlwater with 416 ml 10 N NaOH; BDH AnalaR #10356.4D; freshly made) and 12μl of 10 mM Quinol (0.055 g in 50 ml water, BDH AnalR #103122E; freshlymade) were added in succession. Quinol is a reducing agent and helps toreduce oxidation of the reagents. Other reducing agents can also beused, for example, dithiothreitol (DTT), mercaptoethanol, quinone(hydroquinone), or other suitable reducing agents. The sample wasoverlaid with 200 μl of mineral oil. The overlaying of mineral oilprevents evaporation and oxidation of the reagents but is not essential.The sample was then incubated overnight at 55° C. Alternatively thesamples can be cycled in a thermal cycler as follows: incubate for about4 hours or overnight as follows: Step 1, 55° C./2 hr cycled in PCRmachine; Step 2, 95° C./2 min. Step 1 can be performed at anytemperature from about 37° C. to about 90° C. and can vary in lengthfrom 5 minutes to 8 hours. Step 2 can be performed at any temperaturefrom about 70° C. to about 99° C. and can vary in length from about 1second to 60 minutes, or longer.

After the treatment with Sodium Metabisulphite, the oil was removed, and1 μl tRNA (20 mg/ml) or 2 μl glycogen were added if the DNAconcentration was low. These additives are optional and can be used toimprove the yield of DNA obtained by co-precitpitating with the targetDNA especially when the DNA is present at low concentrations. The use ofadditives as carrier for more efficient precipitation of nucleic acidsis generally desired when the amount nucleic acid is <0.5 μg.

An isopropanol cleanup treatment was performed as follows: 800 μl ofwater were added to the sample, mixed and then 1 ml isopropanol wasadded. The water or buffer reduces the concentration of the bisulphitesalt in the reaction vessel to a level at which the salt will notprecipitate along with the target nucleic acid of interest the dilutionis generally about ¼ to 1/1000 so long as the salt concentration isdiluted below a desired range, as disclosed herein.

The sample was mixed again and left at 4° C. for a minimum of 5 minutes.The sample was spun in a microfuge for 10-15 minutes and the pellet waswashed 2× with 70% ETOH, vortexing each time. This washing treatmentremoves any residual salts that precipitated with the nucleic acids.

The pellet was allowed to dry and then resuspended in a suitable volumeof T/E (10 mM Tris/0.1 mM EDTA) pH 7.0-12.5 such as 50 μl. Buffer at pH10.5 has been found to be particularly effective. The sample wasincubated at 37° C. to 95° C. for 1 min to 96 hr, as needed to suspendthe nucleic acids.

Amplification

PCR amplifications were performed in 25 μl reaction mixtures containing2 μl of bisulphite-treated genomic DNA, using the Promega PCR mastermix, 6 ng/μl of each of the primers. Strand-specific nested primers areused for amplification. 1st round PCR amplifications were carried outusing PCR primers 1 and 4 (see below). Following 1st roundamplification, 1 μl of the amplified material was transferred to 2ndround PCR premixes containing PCR primers 2 and 3 and amplified aspreviously described. Samples of PCR products were amplified in aThermoHybaid PX2 thermal cycler under the conditions: 1 cycle of 95° C.for 4 minutes, followed by 30 cycles of 95° C. for 1 minute, 50° C. for2 minutes and 72° C. for 2 minutes; 1 cycle of 72° C. for 10 minutes.

A representation of the fully nested PCR approach is shown below:

Multiplex Amplification

One μl of bisulphite treated DNA is added to the following components ina 25 μl 20 reaction volume, x1 Qiagen multiplex master mix, 5-100 ng ofeach 1st round INA or oligonucleotide primer 1.5-4.0 mM MgSO4, 400 μM ofeach dNTP and 0.5-2 units of the polymerase mixture. The components arethen cycled in a hot lid thermal cycler as follows. Typically there canbe up to 200 individual primer sequences in each amplification reaction:

Step 1, 94° C. 15 minute 1 cycle

Step 2; 94° C. 1 minute; 50° C. 3 minutes 35 cycles; 68° C. 3 minutes.

Step 3 68° C. 10 minutes 1 cycle

A second round amplification is then performed on a 1 μl aliquot of thefirst round amplification that is transferred to a second round reactiontube containing the enzyme reaction mix and appropriate second roundprimers. Cycling is then performed as above.

HGS ‘Complexity-Reduced’ Primers and Probes

Any suitable PCR primers or probes can be used for the present inventionas well as specially designed primers and probes for non-PCRamplification involving isothermal amplification methodologies. A primeror probe typically has a complementary sequence to a sequence which willbe amplified. Primers or probes are typically oligonucleotides but canbe nucleotide analogues such as INAs. Primers to the ‘top’ and ‘bottom’strands will differ in sequence.

Probes and Primers

A probe or primer may be any suitable nucleic acid molecule or nucleicacid analogue. Examples include, but not limited to, DNA, RNA, lockednucleic acid (LNA), peptide nucleic acid (PNA), MNA, altritol nucleicacid (ANA), hexitol nucleic acid (HNA), intercalating nucleic acid(INA), cyclohexanyl nucleic acid (CNA) and mixtures thereof and hybridsthereof, as well as phosphorous atom modifications thereof, such as butnot limited to phosphorothioates, methyl phospholates, phosphoramidites,phosphorodithiates, phosphoroselenoates, phosphotriesters andphosphoboranoates. Non-naturally occurring nucleotides include, but notlimited to the nucleotides comprised within DNA, RNA, PNA, INA, HNA,MNA, ANA, LNA, CNA, CeNA, TNA, (2′-NH)-TNA, (3′-NH)-TNA, α-L-Ribo-LNA,α-L-Xylo-LNA, β-D-Xylo-LNA, α-D-Ribo-LNA, [3.2.1]-LNA, Bicyclco-DNA,6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA,Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA,β-D-Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA, 2′-R-RNA, α-L-RNA orα-D-RNA, β-D-RNA. In addition non-phosphorous containing compounds maybe used for linking to nucleotides such as but not limited tomethyliminomethyl, formacetate, thioformacetate and linking groupscomprising amides. In particular nucleic acids and nucleic acidanalogues may comprise one or more intercalator pseudonucleotides.

The probes or primers can be DNA or DNA oligonucleotides containing oneor more internal IPNs forming INA.

Detection Methods

Numerous possible detection systems exist to determine the status of thedesired sample. Detection systems include, but not limited to:

I. Hybridization of appropriately labelled DNA to a microarray typedevice which could select for 10->200,000 individual components. Thearrays could be composed of either INAs, PNAs or nucleotide or modifiednucleotides arrays onto any suitable solid surface such as glass,plastic, mica, nylon, bead, magnetic bead, fluorescent bead or membrane;

II. Southern blot type detection systems;

III. Standard PCR detection systems such as agarose gel, fluorescentread outs such as Genescan analysis. Sandwich hybridisation assays, DNAstaining reagents such as ethidium bromide, Sybr green, antibodydetection, ELISA plate reader type devices, fluorinieter devices; IV.Real-Time PCR quantitation of specific or multiple genomic amplifiedfragments or any variation on that;

V. Any of the detection systems outlined in the WO 2004/065625 such asfluorescent beads, enzyme conjugates, radioactive beads and the like;

VI. Any other detection system utilizing an amplification step such asligase chain reaction or Isothermal DNA amplification technologies suchas Strand Displacement Amplification (SDA).

VII. Biosensor technology such as U.S. Pat. No. 6,426,231 and U.S. Pat.No. 6,916,665 in the name of The Texas A&M University System, U.S. Pat.No. 6,824,659 in the name of University of Massachusetts, incorporatedherein by reference, would be suitable for the present invention.

Intercalating Nucleic Acids

Intercalating nucleic acids (INA) are non-naturally occurringpolynucleotides which can hybridize to nucleic acids (DNA and RNA) withsequence specificity. INA are candidates as alternatives/substitutes tonucleic acid probes and primers in probe-, or primer-based,hybridization assays because they exhibit several desirable properties.INAs are polymers which hybridize to nucleic acids to form hybrids whichare more thermodynamically stable than a corresponding naturallyoccurring nucleic acid/nucleic acid complex. They are not substrates forthe enzymes which are known to degrade peptides or nucleic acids.Therefore, INAs should be more stable in biological samples, as well ashaving a longer shelf-life than naturally occurring nucleic acidfragments. Unlike nucleic acid hybridization which is very dependent onionic strength, the hybridization of an INA with a nucleic acid isfairly independent of ionic strength and is favoured at low ionicstrength under conditions which strongly disfavour the hybridization ofnaturally occurring nucleic acid to nucleic acid. The binding strengthof INA is dependent on the number of intercalating groups engineeredinto the molecule as well as the usual interactions from hydrogenbonding between bases stacked in a specific fashion in a double strandedstructure. Sequence discrimination is more efficient for INA recognizingDNA than for DNA recognizing DNA.

Preferably, the INA is the phosphoramidite of(S)-1-O-(4,4′-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)-glycerol.

INAs are synthesized by adaptation of standard oligonucleotide synthesisprocedures in a format which is commercially available. Full definitionof INAs and their synthesis can be found in WO 03/051901, WO 03/052132,WO 03/052133 and WO 03/052134 (Human Genetic Signatures Pty Ltd)incorporated herein by reference.

There are indeed many differences between INA probes and primers andstandard nucleic acid probes and primers. These differences can beconveniently broken down into biological, structural, andphysico-chemical differences. As discussed above and below, thesebiological, structural, and physico-chemical differences may lead tounpredictable results when attempting to use INA probes and primers inapplications were nucleic acids have typically been employed. Thisnon-equivalency of differing compositions is often observed in thechemical arts.

With regard to biological differences, nucleic acids are biologicalmaterials that play a central role in the life of living species asagents of genetic transmission and expression. Their in vivo propertiesare fairly well understood. INA, however, is a recently developedtotally artificial molecule, conceived in the minds of chemists and madeusing synthetic organic chemistry. It has no known biological function.

Structurally, INAs also differ dramatically from nucleic acids. Althoughboth can employ common nucleobases (A, C, G, T, and U), the compositionof these molecules is structurally diverse. The backbones of RNA, DNAand INA are composed of repeating phosphodiester ribose and2-deoxyribose units. INA differs from DNA or RNA in having one or morelarge flat molecules attached via a linker molecule(s) to the polymer.The flat molecules intercalate between bases in the complementary DNAstand opposite the INA in a double stranded structure.

The physico/chemical differences between INA and DNA or RNA are alsosubstantial. INA binds to complementary DNA more rapidly than nucleicacid probes or primers bind to the same target sequence. Unlike DNA orRNA fragments, INA bind poorly to RNA unless the intercalating groupsare located in terminal positions. Because of the strong interactionsbetween the intercalating groups and bases on the complementary DNAstrand, the stability of the INA/DNA complex is higher than that of ananalogous DNA/DNA or RNA/DNA complex.

Unlike other nucleic acids such as DNA or RNA fragments or PNA, INAs donot exhibit self aggregation or binding properties.

In summary, as INAs hybridize to nucleic acids with sequencespecificity, INAs are useful candidates for developing probe-, orprimer-based assays and are particularly adapted for kits and screeningassays. INA probes and primers, however, are not the equivalent ofnucleic acid probes and primers. Consequently, any method, kits orcompositions which could improve the specificity, sensitivity andreliability of probe-, or primer-based assays would be useful in thedetection, analysis and quantitation of DNA containing samples. INAshave the necessary properties for this purpose.

HPV and Cancer Genomic Markers

Using clinical samples and cell lines, the present inventors have lookedat methylation patterns in regulatory regions of nearly 400 genes andhave found over 60 genomic markers that have methylation changes when ina cancerous state together with the presence of HPV. Examples include,but not limited to, one or more of the following genomic regions within,or near to, the transcription units, (genes) denoted CD14, ENDRB, HIC,RARB1, PGR, SFRS8, TMSB10, ABCG2, MFNG, LAMR1, RAGE, ABL1, CRBP, GPR37,HRK, RARA, SYK, ECE1, MME, TEM, NF2, XIAPHSX11, RARRES1, FLI1, HTLF,LDHB, RB1, TGD, CDK4, MMP14, RAB32, BARD1, NF1, LIM2, MMP2, DAB2, BMP6,CDKN1C, DAB21P, LMNB1, MMP28, HAI2, SOCS1, HIC2, MSH6, RIN2, HMGA1, JUN,S100P*, SRF, VDR, DKK3, KRAS2, PLAU, TNFRSF10B, CDH1, MAC30, DDB2, PAX6,AXL, EIF4A2, SLIT2, RECK, TERC, GATA5, STAT1.

Disease States

The present inventors have used HPV and methylation of selected humangenomic regions in cervical cancer as an example of demonstrating thepresent invention. It will be appreciated that other states such asvarious forms of dementia, (Alzheimers), may have an infectiouscomponent such as Herpes simplex virus type 1, (2004, Neurobiology ofAging, 25, 619-627; Itzhaki, R. F., et al.); the clinical progressionand viral load associated with coronavirus-associated SARS pneumonia,(2003, The Lancet, 361, 1767-1772, Peiris, J. S. M. m et al.; humanimmunodeficiency virus HIV, and immune system compromise and severalviral-based hemorraghic fevers, (2004, Nature Medicine, 10, 570-576,Weiss, R. A., et al.,); the viruses of the family Paramyxoviridae whichinclude Nipah virus, parainfluenza and Mumps and are associated withvarious respiratory illnesses, mumps, meningitis, pancreatitis,encephalitis and measles. Nipah virus was only recognised first in 1999and it causes fatal encephalitis in 70% of infected patients and has anextremely broad host range including humans, dogs, cats, pigs, horses,hamsters, bats and guinea pigs. It is a critical threat to global healthand economies (2005, Nature, 436, 401-405; Negrete, O. A., et al,);viruses of the Flaviviridae, which include Dengue, Yellow Fever,Hepatitis C and G and are associated with encephalitis, hepatitis andshock syndrome. Hep C for example, is a major cause of chronic liverdisease with over 170 million individuals infected worldwide and with noavailable vaccine, (2005, Science, 309, 623-626; Lindenbach, B. D., etal,); and finally viruses of the family Herpesviridae, which includehuman herpesvirus 1 through 8. These viruses can give rise to oralinfections, ulceration of the cornea, genital tract infections,meningitis, chickenpox, pneumonia, shingles, cytomegaloviralmononucleosis and encephalitis. Human Cytomegalovirus causes severe andfatal diseases in immunocompromised individuals, including organtransplant individuals, (2003, Nature, 424, 456-461; Wang, X., et al)would also be candidates for the present invention in humans as well asvarious animals as progression of the Health State can be monitored by acombination of viral presence and methylation state of the DNA of thehost. Similar examples are available for plant viruses and viroids.

Examples HPV

To demonstrate the present invention, HPV and cervical cancer will beused in the following examples. It will be appreciated that otherviruses and disease states, as outlined above can also be assayed by thepresent invention.

To reiterate the foundations on which the present inventors have basedtheir bioinformatic analyses in silico, the standard HPV type utilizedherein for reference purposes is HPV16 of the Family Papovaviridae,Genus Papillomavirus, originally designated as such by the InternationalCommittee on Taxonomy of Viruses, ICTV, (1993, Van Rast, M. A., et al.,Papillomavirus Rep, 4, 61-65; see also, 1998 Southern, S. A. andHerrington, C. S. Sex. Transm. Inf. 74, 101-109), although taxonomicupgrades to the Papillomaviridae are sometimes used interchangeably inthe prior art. To avoid ambiguity, the present inventors used the fullysequenced 7904 base pair genome of HPV16 as a standard comparator(National Center for Biotechnology Information, NCBI locus NC_(—)001526;version NC_(—)001526.1; GI:9627100; references, Medline, 91162763 and85246220; PubMed 1848319 and 2990099).

In addition, the present inventors used the fully sequenced genomes ofthe so called high-risk HPV types 16, 18, 45 and 56 with NCBI accessionnumbers of NC-001526, NC-001357, NC-001590 and NC-001594 respectively.

The present inventors used the fully sequenced genomes of the so calledmedium risk HPV types 30, 31, 33, 35, 39, 51, 52, 58 and 66 with NCBIaccession numbers of NC-001585, NC-001527, NC-001528, NC-001529,NC-001535, NC-001533, NC-001592, NC-001443 and NC-001695 respectively.

The present inventors used use the fully sequenced genomes of the socalled low risk HPV types 6, 11, 42, 43, 44, 53, 54 and 55 with NCBIaccession numbers of NC-00904, NC-001525, NC-001534, NC-005349,NC-001689, NC-001593, NC-001676 and NC-001692 respectively.

As the present inventors have demonstrated, the detection of humanpapilloma viral DNA in various clinical samples via conventional DNAtests is hampered by a number of technical, methodological and clinicalproblems. The present invention provides a solution to many of thedifficulties encountered in the prior art, since the bisulphiteconversion of HPV DNA reduces the complexity of the HPV derivativesequence pool. This complexity-reduction allows for a more efficientinitial screening of the different HPV types within a sample and hencefor a more appropriate and accurate interface with the clinical data.

Examples of the detection of HPV in a sample using methods developed bythe applicant can be found in WO 2006/066353 (PCT/AU2005/001963) in thename of Human Genetic Signatures Pty Ltd, incorporated herein byreference.

FIG. 1 shows the results of testing various normal individuals, as wellas those with High Grade Squamous Intraepithelial Lesions, and a cellline for the presence of HPV. The data provided complement the data ofFIG. 1 from patients who have been tested for HPV as well as themethylation status of 400 human genomic regions.

TABLE 2 Expected fragment sizes in base pairs of amplified nucleic acidproducts generated from different HPV derivatives of the three majorrisk types. HPV Risk Category PCR product band size (bp) High SizeMedium Size Low Size HPV16 205 HPV30 302 HPV6 353 HPV18 231 HPV31 216HPV11 268 HPV45 217 HPV33 234 HPV42 228 HPV56 272 HPV35 351 HPV43 251HPV39 230 HPV44 246 HPV51 251 HPV53 207 HPV52 259 HPV54 248 HPV58 182HPV55 303 HPV66 255

The Dual Presence of HPV and the Methylation Status of Various HumanGenomic Regions

The experiments were devised to demonstrate that the presence of HPVtogether with the methylation status of specific genomic regions is amore powerful prognostic indicator of health state than eithercharacteristic alone. In this manner, a large number of genomic regionshave been assayed for their methylation status in samples fromindividuals whose cervical cytology is normal; individuals whosecervical cytology is characteristic of High Grade IntraepithelialLesions, and an individual whose cervical cytology is normal as regardscancerous indicators of cell morphology, but who is inferred to bepositive for HPV on the basis of cytological characteristics.

FIG. 1A shows that a HeLa cell line, two patients exhibiting HSIL asdetermined by a pathologist, (denoted HSIL-1 and HSIL-2), and onepatient with normal cervical morphology but with the presence of HPVinferred from the pathological phenotype (denoted HPV+Nor) were positivefor the presence of HPV as determined by appropriate PCR amplificationtechnology. Two patients with normal cervical morphology (denotedNormal-1 and Normal-2) were molecularly negative for the presence ofHigh-Risk HPV types.

FIG. 1B shows the presence of a band, (an amplicon), in the HeLa cellline, (panel 1), and this contains HPV18 type DNA sequences. The twoHSIL samples, (panels 2 and #), contain HPV16 as assayed molecularly.The cervical tissue which was determined to be morphologically normal bya pathologist, but some cells of which had characteristic cytoplasmicfeatures that were indicative of HPV infection, was found to be positivefor HPV type 82, (panel 4).

These clinical samples were then tested at each genomic locus for thepresence of methylation, (denoted pos), or non-methylation, (denotedneg). The crucial prognostic indicators are, for example, when patientswith normal pathology have a non-methylated locus whereas patients withHigh grade Intraepithelial Lesions have methylation at the same region;in short, the associated gene region has been silenced in theprogression to the cancerous state. (The converse of course applies.Individuals with normal cytology may have a locus that is methylated ina particular cell type, and that locus becomes unmethylated in thecancerous state).

Since methylation of a given genomic region varies with cell type, somegenomic regions will of necessity be uninformative as regardsprogression to the cancerous state in that particular cell type. Theseregions will either be completely unmethylated in all samples, orcompletely methylated in all clinical samples.

Table 3A and 3B show the HPV status of a number of clinical samples, aswell as that of a HeLa cell line, and the methylation status of 53individual human genomic regions.

As can be seen from Tables 3A and 3B both the HeLa cervical cancer cellline and the High grade Squamous Inter-epithelial Lesions (HSIL) werepositive for the presence of HPV DNA as determined molecularly. Onenon-cancerous cervical tissue sample, but with an HPV infection asinferred pathologically, (denoted HPV+Nor) was also found to be positivefor the presence of HPV. The two normal cervical tissue samples fromdifferent individuals were found to be negative for the presence of HPVDNA sequences as determined molecularly.

Each of the above regions was PCR amplified and the resulting ampliconsdigested with appropriate restriction enzymes (and/or sequenced) todetermine the methylation status of that particular genomic region(Tables 3A and 3B).

First, there are genomic regions that are uninformative as regards beingprognostic indicators. These are unmethylated in all samples, (13genomic regions ABCG2 to VHL), or methylated in all samples, (4 genomicregions, CD34, MAGEA2, MAGEA3 and MINT31). Of particular interest fromthe results in Tables 3A and 3B are genomic regions denoted CD14, ENDRB,HIC and RARB. All of these regions were methylated in the HeLa cervicalcancer cell line and both HSIL samples. Interestingly, none of theseregions was methylated in either the normal cervical tissues tested orin the non-cancerous cervical tissue sample infected with HPV.

Finally, 32 genomic regions ANAX7 through TNFRS10B, show variablemethylation patterns between normal individuals and those with HSIL.This variation very likely reflects a mixture of the genetic backgroundof individual patients and the differing stabilities of the methylationstatus at individual loci of the human genome.

The results indicate that although the presence of HPV has been detectedin almost all HSIL and cervical cancers, the presence of HPV alone isnot a reliable indicator of high grade abnormalities of the cervix.However, when the presence of HPV is linked with a change in themethylation profile of cervical DNA samples this gives a much betterprognostic indicator of disease state.

TABLE 3A

TABLE 3B

ND = not determined

In addition to methylation of genomic regions CD14, ENDRB, HIC and RARBthe present inventors have also identified a further 62 DNA genomicregions that show a similar methylation profile as CD14, ENDRB, HIC andRARB in HSIL samples but not in normal cervical tissue or normalcervical tissue infected with HPV from a panel of 384 candidate genes.These markers are listed in Table 4.

TABLE 4 Genomic regions tested as potential markers together with theircorresponding Genbank numbers. Genomic Genbank Genomic Genbank GenomicGenbank Genomic Genbank PGR AY525610 NF2 AF165426 BMP6 AF083030 KRAS2M30539 SFRS8 XIAPHSX11 CDKN1C U48869 PLAU X02419 TMSB10 M92383 RARRES1DAB2IP AL357936 TNFRSF10B AB054004 ABCG2 FLI1 AF275879 LMNB1 L37737 CDH1MFNG HTLF AC091485 MMP28 AF336346 MAC30 LAMR1 LDHB X13794 HAI2 DDB2 RAGERB1 AF551763 SOCS1 DQ086801 PAX6 ABL1 U07563 TGD AF545435 HIC2 AXL CRBPX07437 CDK4 AF507942 MSH6 AY082894 EIF4A2 GPR37 AC004925 MMP14 AY795074RIN2 AL049538 SLIT2 HRK RAB32 AL133539 HMGA1 L17131 RECK RARA BARD1AC016708 JUN J04111 TERC SYK AL354862 NF1 S100P* GATA5 ECE1 AL031005LIM2 AF305941 SRF AL133375 STAT1 MME M26605 MMP2 AY738117 VDR AY342401TEM AL035608 DAB2 U41111 DKK3 AB035182

FIG. 2 shows the results of PCR amplification on 36 LBC samples for bothhuman genomic DNA and HPV DNA using the HGS HR-HPV DNA purification anddetection kit. As can be seen from the results using this method it ispossible to assay for the presence of both human genomic changes and thepresence of absence of virus simultaneously.

FIG. 3 shows a representative gel of normal cervical samples amplifiedat 16 different genomic loci, digested with a combination of BstU1 andTaqαI restriction endonuclease, and electrophoresed on an agarose gel.DNA was extracted from liquid based cytology specimens (numbers 28 and29 here), sodium bisulphite modified and amplified with nested primersto genes identified for further analysis. These genes were, from left toright, 1) TEM 2) MME 3) ECE14) SYK 5) RARA 6) HRK 7) GPR37 8) CRBP 9)ABL1 10) RAGE 11) LAMR1 12) MFNG 13) ABCG2 14) TMSBIO 15) SFRS8 and 16)PGR.

The pathology and HPV infection profile of the samples were assessed byan expert pathologist. When the CpG dinucleotides within the ampliconwere unmethylated, bisulphite modification converts the unmethylatedcytosines to a uracil base and after amplification to a thymine base.The unmethylated CpG dinucleotide and the restriction site, is notretained and therefore not recognized by BstU1 (recognises CGCG, whichwill be converted to TGTG after amplification if the sample isunmethylated but will remain CGCG if the sample contains methylatedsequences) or TaqαI (recognises TCGA, which will be converted to TTGAafter amplification if the sample is unmethylated but will remain TCGAif the sample contains methylated sequences) endonucleases. Detection ofa single band, representing undigested PCR product, therefore impliesthat the CpG dinucleotide within the amplicon is unmethylated.Methylated CpG dinucleotide is resistant to bisulphite modification sorestriction sites recognized by the restriction endonucleases areretained. Consequently the PCR products are digested into multiplefragments (as indicated by an asterisk) depending on the number of CpGsites available in the amplicon. Detection of an empty lane suggeststhat the PCR reaction was unsuccessful.

Few multiple bands were detected at these 16 loci. Representativeresults from this assay are tabulated in Table 5.

TABLE 5 Methylation profiles for DNA samples extracted frompathologically normal cervical samples. Sample ID: C18 C19 C22 C42 C13C14 C24 C25 C26 C28 C29 Norm Norm Norm Norm Norm Norm Norm Norm NormNorm Norm HPV HPV HPV HPV ABCG2 U U U U U U U U U U M MFNG M M U U U U UM M M M LAMR1 U U U U U U U U U M U HRK F U U U U U U U U U U HSX1APF1 UU U U U U U U U M F RARRES1 U U U U U U U U U M M FLI1 U U U U U U U U UU U LDHB U F U U U U U U U U F CDK4 U U U U U U U U U U U MMP14 U F U UU U U U U U C DAB2 U U U U U U U U U U U SOCS1 U U U U U U U F U M UHIC2 U U U U U U U U U U U PLAU U F F U U F U F U U M EIF4A2 U U F F U UU U U U U SLIT2 U U U M M U F M U M U RECK U M U U U U U M U M M TERC FU U U U U F F U M F GATA5 M M M M M M M M U M M STAT1 U U U U U U U U UU U HPV Uni − − − − − − − + − − − HmGST U U U U U U U U U U U

The methylation profile of a representative number of pathologicallynormal cervical samples (NORM), some of which were likely to have a HPVinfection (NORM HPV). DNA was extracted from a liquid based cytologysample, sodium bisulphite modified and amplified at 384 different genes.The amplicons were digested with restriction enzymes and electrophoresedon an agarose gel. Detection of a single product (an undigested product)indicates that the sample is unmethylated (U) at the promoter region ofthe gene being interrogated. Detection of multiple bands (a digestedproduct) implies that the sample is methylated (M) at the promoter ofthe gene being interrogated. The lack of a band at a specific loci (asdenoted by “F”) infers that the PCR reaction was unsuccessful.

The presence (+) or absence (−) of high and medium risk papilloma viralDNA was also listed. Primers that amplified both the human and mouseGST-P1 gene (HmGST) was included as a control for the PCR reaction.Detection of a band implies that DNA was converted and available foramplification.

FIG. 4 shows a representative gel of tumour samples amplified at 16different genomic loci, digested with a combination of BstU1 and TaqαIrestriction endonuclease and electrophoresed on an agarose gel. DNA wasextracted from liquid based cytology specimens (numbers 82,83, 84, 94,95 and 96 here), sodium bisulphite modified and amplified with nestedprimers to genes identified for further analysis. These genes were, fromleft to right, 1) PGR 2) SFRS8 3) TMSBIO 4) ABCG2 5) MFNG 6) LAMR1 7)RAGE 8) ABL1 9) CRBP 10) GPR37 11) HRK 12) RARA 13) SYK 14) ECE1 15) MMEand 16) TEM.

The pathology and HPV infection profile of the samples were assessed byan expert pathologist. When the CpG dinucleotides within the ampliconare unmethylated, bisulphite modification converts the unmethylatedcytosines to a uracil base. The unmethylated CpG dinucleotide, and therestriction site, is not retained and therefore not recognized by BstU1or TaqαI endonucleases. Detection of a single band, representingundigested PCR product, therefore implies that the CpG dinucleotidewithin the amplicon is unmethylated. Methylated CpG dinucleotide isresistant to bisulphite modification so restriction sites recognized bythe restriction endonucleases are retained. Consequently the PCRproducts are digested into multiple fragments (as indicated by anasterisk) depending on the number of CpG sites available in theamplicon. Detection of an empty lane suggests that the PCR reaction wasunsuccessful.

A greater proportion of genes were methylated in tumour samples comparedto normal samples (see FIG. 4). Representative results from this assayare tabulated in Table 6.

In Table 6, the methylation profile for a representative number ofpathologically abnormal cervical samples (as assessed by an expertpathologist). Samples were classified based on the presence of humanpapillomavirus (HPV) and the types of lesions (Low-grade LG, Low-gradeLG with the papillomavirus LG HPV, Previous high-grade Pr HG, High-gradeHG, High grade with a carcinoma in situ 3 component, andcarcinoma-in-situ 3 lesion CIN3).

DNA was extracted from a liquid based cytology sample, sodium bisulphitemodified and amplified at 384 different genes. The amplicons weredigested with restriction enzymes and electrophoresed on an agarose gel.Detection of a single product (an undigested product) is representativeof a sample that is unmethylated (U) at the promoter region of the genebeing interrogated. Detection of multiple bands (a digested product) isrepresentative of a sample that is methylated (M) at the promoter of thegene being interrogated. The lack of a band at a specific loci (asdenoted by “F”) infers that the PCR reaction was unsuccessful.

TABLE 6 Methylation profiles for DNA samples extracted from cervicalsamples with unfavourable pathology C61 C71 C82 C53 C56 C68 C83 C85 LGLG LG C67 C74 C84 Pr Pr HPV HPV HPV HPV HPV HPV LG LG LG HG HG ABCG2 U MU U M U U U U M M MFNG M M M U F U U F U M M LAMR1 M M U U U U U U U M MHRK U U U F U U U M M U U HSX1APF1 U M U F F U M F U U U RARRES1 U M F UF U U U M M M FLI1 U M U F U U U F U U M LDHB U M M F U M U M M M M CDK4U M F F F U M U M M M MMP14 M M U U U M U F U M M DAB2 M M U U F M U U MU U SOCS1 U M U U M U F M U U M HIC2 U M U U M U U U U U U PLAU M M F FM M F F U U F EIF4A2 M M U U F M M U M U U SLIT2 U M U U U U U U U U URECK M M U U F M M F M F M TERC F F F F F F F F F U U GATA5 M M U U U MM M M U M STAT1 U M U U U U F U U U U HPV Uni − + + + + + + − − − −HmGST U U U U U U U U U U U C62 C93 C94 C96 Pr C50 C58 C89 HG HG HG C4C65 HG HG HG HG CIN3 CIN3 CIN3 CIN3 CIN3 C95 ABCG2 U M U M M M U U U UMFNG M M F U M M M M U M LAMR1 F U U U F U M U U M HRK U U U U U U M U MU HSX1APF1 F M U U U U U M F U RARRES1 M M M U U M M U U M FLI1 F U M UU U U U U M LDHB U U M M M M U U F M CDK4 F M U F U M M F M U MMP14 U UU U U M M F U U DAB2 F U U M U U U M U M SOCS1 F U F U U U U U U U HIC2M U U U M U U U U U PLAU M U U U U F U NT F U EIF4A2 F U U M M M U NT MM SLIT2 F U U F M M M NT U U RECK M U U U U U U NT U M TERC M U F F F FF NT F F GATA5 M M U M U M M NT M M STAT1 F U U U U U U NT U U HPV Uni +− − − − − + NT − + HmGST U U U U U U U NT U U

TABLE 7 List of patient pathology Sample No Sample Details C1 HPV C2 CaEndomet C3 HG CIN3 C4 CIN3 Cx C5 AC/AIS C6 HPV C7 HPV C8 NORM C9 NORMC10 NORM C11 NORM C12 NORM C13 NORM C14 NORM C15 NORM C16 NORM C17 NORMC18 HPV NORM C19 HPV NORM C20 NORM C21 HPV NORM C22 HPV NORM C23 NORMC24 NORM C25 NORM C26 NORM C27 NORM C28 NORM C29 NORM C30 NORM C31 NORMC32 NORM C33 NORM C34 NORM C35 NORM C36 NORM C37 NORM C38 NORM C39 NORMC40 NORM C41 NORM C42 HPV NORM C43 NORM C44 NORM C45 NORM C46 NORM C47NORM C48 NORM C49 HG C50 HG C51 Pr HG C52 HG CIN3 C53 Pr HG C54 HG CxC55 SSC Cx C56 Pr HG C57 AC Cx C58 HG C59 HG Cx C60 HPV C61 LG HPV C62Pr HG C63 HG C64 CIN1 HPV C65 CIN3 C66 HPV C67 LG C68 HPV C69 HPV C70INC HG? C71 LG HPV C72 HPV C73 HPV C74 LG C75 HPV C76 HPV C77 HPV C78SCC C79 SCC C80 HPV C81 INC HG C82 HPV LG C83 HPV C84 LG C85 HPV C86 HPVLG C87 HPV C88 HPV C89 HG C90 HG C91 HG C92 HG C93 HG CIN3 C94 HG CIN3C95 Unknown C96 HG CIN3

Table 7 shows the pathology of samples investigated at 64 differentgenomic loci for DNA methylation profile.

Liquid Based cytology samples for 96 patients with pre-determinedpathology were extracted for DNA. The associated pathology of thesamples, pertaining to the type of pathology, the degree of invasion andthe presence or absence of human papillomavirus infection, were assessedby an expert pathologist.

Samples were labelled as cytologically normal (NORM), with a likely HPVinfection (HPV), cytologically normal with a likely HPV infection (HPVNORM), as low grade lesion (LG), as a low grade lesion with HPVinfection (LG HPV), as high grade lesion (HG), as increased high-gradelesion (INC HG), as a previous high grade lesions (Pr HG), as acarcinoma in-situ 1 lesion with a likely HPV infection, as a high Gradecarcinoma in-situ 3 lesion (HG CIN3), as high grade carcinoma (HG Cx),as squamous cell carcinoma (SCC) as a squamous cell carcinoma/cancer(SCC Cx), as an adenocarcinoma (AC Cx), as an endometrial cancer (CaEndomet), as an adenocarcinoma/adenocarcinoma in-situ (AC/AIS).

Table 8 shows the genes or genomic regions found by the presentinventors to be suitable indicators for disease states by the presentinvention. It will be appreciated that a person skilled in the art coulddevise suitable primers or probes to detect changes in one or more ofthese genes or genomic region is accociation with detecting the presenceof viral nucleic acid.

TABLE 8 Disease genes or genomic regions suitable for the presentinvention ABCB1 ABCG2 ABL1(R1) ABL1(R2) ABL2 ABO ADAM23 ADAMT58 AKT1ALOX5 ALX3 AMACR ANXA7 APAF1 APC APO1 APP AR ARHI ARNT ASC ATM AXIN1 AXLAXUD1 BAD BARD1 BCR BDH BENE BIK BIN1 BIRC5 BLM BMP2 BMP6 BMPR1A BRAFBRCA1 BRCA2 CASPASE-8 CAV1 CBFA2T3 CBFB CBLC CCNA1(cycal) CCND2 CCND3CD14 CD34 CD44 CD9 CDC20 CDH1 CDH13 CDK10 CDK4 CDKN1A CDKN1B(p27)CDKN1C(p57) CDKN2A CDKN2A-V2 CDKN2B CDKN2C CDKN2D CDX1 CDX-2 CFTR CGRPCHFR CLDN7 CMYB CNTN2 COPEB COX6C CRBP CREBBP CRK CSPG2 CTNNB1 CX26CXCL-2 DAB2 DAB2IP DAPK1 DAPK2 DBCCR1 DCCR DCK DDB2 DKK1 DKK3 DLC1 DLK1DNAJB9 DNMT1 DRG1 DSC3 DUX4 E2F1 ECE1 EDNRB EFNA4 EGFR EGR3 EIF4A2 ELAC2ELK1 ENO3 EP300 EPB41L3 EPHA1 EPHA8 EPO ERBB2 ERBB4 ERCC4 ESR2 ESR-ALPHAETS1 EZH2LONG FANCF FASN FBP FEZ1 FGF4 FLI1 FLT1 FOLH1 FOS FOSB FRA2FRAT1 FRAT2 FXYD5 GADD45G GAPD GATA3 GATA4 GATA5 GATA6 GLTSCR1 GNA13GNAI1 GNAS GP9 GPC3 GPR37 GPS1 GRB10 GROS1 GSK3B GSN GSTP1 GUSA H19AHAI2 HIC-1 HIC-2 HLAG HMGA1 HOXA11 HOXA13 HOXA5 HOXB5 HOXD13 HOXD8 HPNHPRT HPRT1 HRAS HRK HSPC070 HSXI1PAF1 HTLF ID1 IGF2 IGFBP7 IL6 ILK ING1INHA IRF7 JUN JUNB JUP KAI-1 K-ALPHA-1 KIT KLF4 KPNA3 KRAS2 LAMA3 LAMC2LAMR1 LATS1 LCK LDHA LDHB LEP1 LIF LIM2 LMNA1 LMNB1 LOX LRP2 LRP6 LTFMAC30 MAD2L1 MADH4 MAGE MAGE-A3 MAL MCC MDGI MDM2 MFNG MGMT MIF MINT1MINT25 MINT31 MLH1 MME MMP14 MMP2 MMP28 MRE11A MSH2 MSH6 MSLN MT1G MT3MUC1 MYB MYC MYCN MYOD1 MYOG1 N33 NCOA4 NDN NF1 NF2 NME1 NNAT NOTCH1 NOVNROB2 NTRK1 OCLN OPCML OXCT P16 PAX3 PAX5 PAX6 PAX7 PCNA PDCD2 PDGFDPENK PGR PIM1 PITX2 PLAU PMS2 PNN POMC POU2AF1 PPARG1 PPM1D PPP2R1BPRKAR1A PRKCDBP PRSS8 PSEN1 PTCH PTEN PTGER3 PTGS2 PTPN6 PTPRO PTTG1IPPVT1 RAB32 RAB3A RAB5A RAD51 RAGE RARA RARB RARRES1 RASSF1 RB1 RBL2 RBM5RECK RFC1 RFX1 RGS19IP1 RIN2 RNASE6PL RUNX3 S100P SAC2 SCGB3A1 SEMA3BSFN SFRP1 SFRP2 SFRS8 SHH SLC26A4 SLC5A5 SLIT2 SLS5A8 SMARCA3 SMARCB1SMARCD3 SMOH SMT3H1 SNCG SNRPN SOCS1 SOD1 SOX4 SPARC SPI7 SRF SRP72 SSX2SSX4 STAT1 STAT2 STAT3 STAT4 STK11 SYK TACSTD TAGLN TAUPE- NUSS TDG TEM1TEM8 TERC TERE1 TERT TES TFF1 TFF2 TFP12 TGFBR2 THBS1 THRB TIG TIMP3 TJPTMEFF2 TMSB10 TNC TNFRSF10B TNFRSF10C TNFRSF6 TOP1 TOP2A TP53 TP53BP2TP73 TPD52 TPM1 TRA1 TRAF4 TSC2 TSHR TWIST VAV VDR VEGF VHL WFDC1 WT1YWHAG

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1-23. (canceled)
 24. An assay for determining a health state of asubject comprising: treating a sample from a subject with an agent thatmodifies unmethylated cytosine to form derivative nucleic acid;providing primers capable of allowing amplification of a desired viralnucleic acid molecule to the derivative nucleic acid; providing primerscapable of allowing amplification of a target genomic nucleic acidmolecule to the derivative nucleic acid; carrying out an amplificationreaction on the derivative nucleic acid; and assaying for the presenceof amplified desired viral nucleic acid and amplified target genomicnucleic acid, wherein presence or absence of one or more amplifiedproducts is indicative of a health state of the subject.
 25. The assayaccording to claim 24, further comprising assaying for the presence ofan amplified nucleic acid product containing the desired virus-specificnucleic acid molecule, wherein detection of the desired virus-specificnucleic acid molecule is indicative of the presence of the virus in thesample.
 26. The assay according to claim 24, further comprising assayingfor the presence of an amplified nucleic acid product containing thetarget nucleic acid molecule, wherein detection of the target nucleicacid molecule is indicative of a genomic or gene state in the sample.27. The assay according to claim 24, wherein the agent modifiesunmethylated cytosine to uracil.
 28. The assay according to claim 27,wherein the agent is bisulfite, acetate or citrate.
 29. The assayaccording to claim 28, wherein the agent is sodium bisulfite.
 30. Theassay according to claim 24, wherein the target genomic nucleic acid isspecific for a gene or genes or regulatory region.
 31. The assayaccording to claim 30, wherein the target has a methylationcharacteristic.
 32. The assay according to claim 31, wherein themethylation characteristic is a methylated or unmethylated region ofgenomic nucleic acid.
 33. The assay according to claim 24, wherein thevirus is implicated in a disease state.
 34. The assay according to claim33, wherein the virus is selected from the group consisting of humanpapilloma viruses, hepatitis viruses, human immunodeficiency virus 1(HIV-1), human immunodeficiency virus 2 (HIV-2), human herpesviruses,retroviruses, polyoma viruses, and adenoviruses.
 35. The assay accordingto claim 24, wherein the genomic target is implicated in a diseasestate.
 36. The assay according to claim 35, wherein the genomic targetis selected from the group consisting of regions of CD14, ENDRB, HIC,RARB1, PGR, SFRS8, TMSB10, ABCG2, MFNG, LAMR1, RAGE, ABL1, CRBP, GPR37,HRK, RARA, SYK, ECE1, MME, TEM, NF2, XIAPHSX11, RARRES1, FLI1, HTLF,LDHB, RB1, TGD, CDK4, MMP14, RAB32, BARD1, NF1, LIM2, MMP2, DAB2, BMP6,CDKN1C, DAB21P, LMNB1, MMP28, HAI2, SOCS1, HIC2, MSH6, RIN2, HMGA1, JUN,S100P*, SRF, VDR, DKK3, KRAS2, PLAU, TNFRSF10B, CDH1, MAC30, DDB2, PAX6,AXL, EIF4A2, SLIT2, RECK, TERC, GATA5, and STAT1.
 37. The assay fordetermining a health state of a subject according to claim 24, furthercomprising: treating a sample from a subject with a bisulfite reagentunder conditions that cause unmethylated cytosines in viral and genomicnucleic acid to be converted to uracil forming derivative viral nucleicacid and derivative genomic nucleic acid; providing primers capable ofbinding to regions of derivative viral nucleic acid to the sample, theprimers being capable of allowing amplification of a desiredviral-specific nucleic acid molecule in the derivative viral nucleicacid; providing primers capable of binding to regions of derivativegenomic nucleic acid to the sample, the primers being capable ofallowing amplification of a desired target genomic-specific nucleic acidmolecule in the derivative genomic nucleic acid; carrying out anamplification reaction on the treated sample; and assaying for thepresence of an amplified viral nucleic acid product and an amplifiedgenomic nucleic acid target, wherein detection of one or both of theproduct and target is indicative of a health state of the subject. 38.The assay according to claim 37, further comprising testing a samplehaving a virus present to determine the type, subtype, variant orgenotype of the virus in the sample.
 39. The assay according to claim 37wherein the disease state is cervical cancer.
 40. The assay according toclaim 38 wherein amplification is carried out by polymerase chainreaction (PCR) or isothermal amplification.
 41. An assay for screeningfor potential cervical cancer in a subject comprising: treating a samplefrom the subject with bisulfite reagent under conditions that causeunmethylated cytosines in human papilloma virus (HPV) and genomicnucleic acid to be converted to uracil to form derivative HPV nucleicacid and derivative genomic nucleic acid; providing primers capable ofbinding to regions of derivative HPV nucleic acid, the primers beingcapable of allowing amplification of a desired HPV-specific nucleic acidmolecule of the derivative HPV nucleic acid; providing primers capableof binding to regions of derivative genomic nucleic acid, the primersbeing capable of allowing amplification of a desired genomic-specificnucleic acid molecule of the derivative genomic nucleic acid; carryingout an amplification reaction on the derivative HPV nucleic acid andderivative genomic nucleic acid; and assaying for the presence of anamplified HPV nucleic acid product and an amplified genomic nucleic acidproduct, wherein detection of one or both products is indicative ofprogression to a cervical cancer state in the subject.
 42. The assayaccording to claim 41, further comprising testing a sample having thepresence of a HPV to determine the type, subtype, variant or genotype ofthe HPV in the sample.
 43. The assay according to claim 42 wherein thesample is selected from the group consisting of swab, biopsy, smear, Papsmear, surface scrape, spatula, and fluid samples, as well as samplesfrom different storage media such as frozen material, paraffin blocks,glass slides, forensic collection systems, and archival material.